NZ791706A - Regulation of gene expression using engineered nucleases - Google Patents
Regulation of gene expression using engineered nucleasesInfo
- Publication number
- NZ791706A NZ791706A NZ791706A NZ79170617A NZ791706A NZ 791706 A NZ791706 A NZ 791706A NZ 791706 A NZ791706 A NZ 791706A NZ 79170617 A NZ79170617 A NZ 79170617A NZ 791706 A NZ791706 A NZ 791706A
- Authority
- NZ
- New Zealand
- Prior art keywords
- cell
- cells
- dna
- sequence
- donor
- Prior art date
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Abstract
The present disclosure is in the field of genome engineering, particularly targeted modification of the genome of a hematopoietic cell.
Description
REGULATION OF GENE EXPRESSION USING ENGINEERED
NUCLEASES
CROSS-REFERENCE TO RELATED ATIONS
The present ation claims the t of U.S. Provisional
Application No. 62/378,978, filed August 24, 2016; U.S. Provisional Application No.
62/443,981, filed January 9, 2017; and U.S. Provisional Application No. 62/545,778,
filed August 15, 2017, the disclosures of which are hereby incorporated by reference
in their entireties. The present application claims divisional status from New Zealand
Application 750938 the entire contents of which are hereby incorporated by cross
reference.
CAL FIELD
The t disclosure is in the field of genome engineering,
particularly targeted modification of the genome of a hematopoietic cell.
BACKGROUND
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. sion of genes is controlled by a highly complex mixture of
general and specific transcriptional regulators that interact with 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, tors and locus control regions (LCRs) (see Matson et al.
(2006) Ann Rev Genome Hum Genet 7: 29-50).
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 can function to regulate expression at large distances from the core
promoter of the targeted gene, and are not dependent on any ic sequence
orientation with t to the promoter. Enhancers can be located several hundred
18984823_1 (GHMatters) P44888NZ01
WO 39440 2017/048397
kilobases upstream or ream of a core promoter region, where they can be
located in an intron sequence, or even beyond the 3’ end of a gene.
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., US. Patent Nos. 9,255,250, 9,200,266, 9,045,763, 9,005,973, 9,150,847,
8,956,828, 8,945,868, 8,703,489, 8,586,526, 6,534,261, 6,599,692, 6,503,717,
6,689,558, 317, 7,262,054, 7,888,121, 7,972,854, 796, 7,951,925,
379, 8,409,861, US. Patent ation Nos. 2003/0232410, 2005/0208489,
026157, 2005/0064474, 2006/0063231, 2008/0159996, 2010/00218264,
2012/0017290,2011/0265198,2013/0137104,2013/0122591,2013/0177983,
2013/0196373, 2015/0056705 and 335708, the disclosures ofwhich are
incorporated by nce in their entireties.
[0006] 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), non-homology directed end capture of donors or repair using a repair
template (homology directed repair or HDR) can result in the knock out of a gene or
2O the insertion of a sequence of interest (targeted ation). See, e.g., US. Patent
Nos. 9,045,763, 9,200,266, 9,005,973, and 8,703,489. These techniques 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 chNA/tracr RNA le guide
RNA’) to guide specific cleavage. Further, targeted nucleases are being ped
based on the Argonaute system (e.g., from T lhermophilus, known as ‘TtAgo’, see
Swarts er a]. (2014) Nature 507(7491): 258-261), which also may have the potential
for uses in genome editing and gene therapy.
Red blood cells , or erythrocytes, are the major cellular
component of blood and account for one quarter of the cells in a human. Mature
RBCs lack a nucleus and many other organelles and are full of hemoglobin, a
metalloprotein that functions to carry oxygen from the lungs to the s 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 (0t)—like globin chains and two beta (B)—like globin chains and 4
heme groups. In , the (1202 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 al in terms of hemoglobin and
RBC stabilization. In a developing fetus, a ent form of hemoglobin, fetal
hemoglobin (HbF), is produced which has a higher g affinity for oxygen than
Hemoglobin A such that oxygen can be delivered to the baby’s system via the
mother’s blood stream. There are two genes that encode fetal globin that are very
similar in sequence and are termed HBGl (also referred to as Ggamma) and HBG2
(Agamma), based on their order of arrangement in the beta globin gene locus. Like
adult hemoglobin, fetal hemoglobin protein contains two 0L globin chains, but in place
of the adult in chains, it has two fetal gamma (y)-globin chains (i.e., fetal
hemoglobin is (123/2). At approximately 30 weeks of gestation, the synthesis of
gamma globin in the fetus starts to drop, while the tion of beta globin
increases. By approximately 10 months of age, the newborn’s hemoglobin is nearly
2O all (1202 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.
c defects in the sequences encoding the hemoglobin chains can
be responsible for a group of diseases known as obinopathies that include
sickle cell anemia and the alpha and beta thalassemias. In the majority of patients with
hemoglobinopathies, the genes encoding gamma globin remain present, but
sion is relatively low due to normal gene repression occurring around
parturition as described above.
[0009] It is estimated that l in 5000 people in the US. have sickle cell disease
(SCD), mostly in people of sub-Saharan Africa descent (Roseff (2009)
Immunohemalologg/ 25(2):67) There appears to be a benefit for heterozygous carriers
of the sickle cell mutation due to protection against malaria, so this trait may have
been positively selected over time, such that it is estimated that in sub-Saharan Africa,
up to 28% of the population has the sickle cell trait (Elguero el al. (2015) PNAS USA
112 (22): 7051). Sickle cell disease is caused by a mutation in the B globin gene as a
consequence of a valine substitution for glutamic acid at amino acid #6 (a GAG to
GTG mutation at the DNA level), where the resultant hemoglobin is referred to as
lobin S” 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 hobic 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
ality or ‘sickling’ of the RBCs, resulting in a loss of cell flexibility. 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.
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
yurea therapy are still unknown, however, and treatment gives unwanted side
effects that lead to low patient compliance, and has le efficacy from patient to
t (Brandow and Panepinto (201 1) Am JHematol 86(9):804-806). 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.
Thalassemias are also es relating to hemoglobin and typically
involve a reduced expression of globin chains. This can occur through mutations in
the regulatory s of the genes or from a mutation in a globin coding sequence
that results in reduced sion or d levels or functional globin protein.
Alpha thalassemias, caused by mutations in the alpha globin locus, are mainly
ated with people of Western Africa and South Asian descent, and may confer
malarial resistance. Beta thalassemia, caused by mutations in the beta globin locus, is
mainly associated with people of Mediterranean descent, typically from Greece and
the coastal areas of Turkey and Italy. In thalassemia minor, only one of the B globin
WO 39440
alleles bears a mutation. Individuals will suffer from microcytic anemia, and
detection usually involves lower than normal mean corpuscular volume (<80fL). The
alleles of subjects with thalassemia minor are BHB or BO/B (where ‘B+’ refers to
alleles that allow some amount of [3 chain formation to occur, ‘B’ refers to wild type B
globin alleles, and ‘BO’ refers to B globin mutations associated with a te
absence of lobin expression). Thalassemia intermedia ts can often
manage a normal life but may need occasional usions, especially at times of
illness or pregnancy, depending on the severity of their . These patient’s
alleles can be B+/B+ or Bo/B+. semia major occurs when both alleles have
thalassemia mutations. This is severely microcytic and hypochromic anemia.
Untreated, it causes anemia, splenomegaly and severe bone deformities and
progresses to death before age 20. Treatment consists of periodic blood usion,
splenectomy for splenomegaly and chelation of transfusion-caused iron overload.
Bone marrow lants 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.
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 expression 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 Nai’l
Acad Sci USA 79(14):4428-31). Subsequent s confirmed the ability of 5-
azacytidine to increase HbF in patients with B-thalassemia and sickle cell disease
(Ley, ei al., (1982) N. Engl. J. Medicine, 307: 1469-1475, and Ley, ei al., (1983)
Blood 62: 370-3 80). In addition, short chain fatty acids (e.g. butyrate and derivatives)
have been shown in experimental systems to increase HbF antoulakis el al.,
(1988) Blood 72(6): 1961-1967). Also, there is a t of the human population
with a condition known as ‘Hereditary Persistence of Fetal Hemoglobin’ (HPFH)
where ed amounts of HbF persist in adulthood (10-40% in HPFH heterozygotes
(see Thein ei a]. (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 sion of HbF can lessen the severity of the disease
(Potoka and Gladwin (2015) Am JPhysiol Lung CellM0] Physio]. 308(4): L314—
L324). 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.
One approach to increase the sion of HbF involves identification
of genes whose products play a role in the regulation of gamma globin expression.
One such gene is BCLl 1A, first identified because of its role in lymphocyte
development. BCLl 1A 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, the ng of the
BCL11A mRNA is pmentally regulated. In embryonic cells, the shorter
BCLl 1A mRNA variants, known as BCLl 1A-S and BCLl 1A-XS are primarily
expressed, while in adult cells, the longer BCLl 1A-L and BCLl 1A-XL mRNA
ts are predominantly expressed. See, Sankaran el 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 . Use of an
inhibitory RNA targeted to the BCLl 1A gene has been proposed (see, e.g., US.
Patent Publication No. 182867) 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.
Targeting of BCLl 1A enhancer sequences es a mechanism for
sing HbF. See, e.g., US. Patent Publication No. 2015/0132269 and PCT
ation No.
a set of genetic variations at the BCLl 1A gene locus that are associated with
increased HbF levels. These variations are a tion of small nucleotide
polymorphisms (SNP) found in non-coding regions of BCLl 1A that on as a
specific, lineage-restricted enhancer region. Further investigation revealed that
this BCL11A enhancer is required in erythroid cells for BCLl 1A expression, but is
not required for its expression in B cells (see Bauer el al. (2013) Science 343 :253-
257). The enhancer region was found within intron 2 of the BCLl 1A gene, and three
areas of DNAseI hypersensitivity (often indicative of a chromatin state that is
associated with tory 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 BCLl 1A. These enhancer regions are
roughly 350 (+55), 550 (+58), and 350 (+62) nucleotides in length (Bauer 2013, ibia’).
When developing a nuclease for use in eutic treatments of
humans, it is essential that the se have the utmost safety characteristics.
cally, the nucleases must have very low levels of off-target cleavage.
Significant numbers of double strand cuts in locations other than the user-specified
target can lead to repression of off-target genes, and in rare instances, the ence
of chromosomal translocations (see Hoban and Bauer (2016) Blood, 127(21):2525-
2535 and Tsang el al. (2017) Nature Methods, in press). Improvements in specificity
can be achieved by eliminating non-specific interactions between the engineered
nuclease and the genomic DNA (see US. Provisional Patent Application Nos.
62/378,978 and 62/443,981).
Thus, there remains a need for additional highly specific methods and
compositions for the alteration of BCLl 1A gene expression, for example to treat
obinopathies such as sickle cell disease and beta thalassemia.
SIHVIMARY
The present invention describes highly specific compositions and
methods for use in gene therapy and genome engineering. Specifically, the methods
and itions described relate to inactivating (e.g., by completely or partially
abolishing its expression) a BCLl 1A gene, for e, a gene that acts as regulator
of one or more additional genes. In ular, the invention describes methods and
compositions for interfering with enhancer function in a BCLl 1A gene to diminish or
knock out its activity in specific cell lineages (e.g., erythroid). Additionally, the
ion provides methods and itions for interfering with BCLl 1A enhancer
functions wherein the enhancer sequences are not located within the coding sequences
of the BCLl 1A gene, and wherein the reagents provided exhibit highly specific
activity. The resulting down-regulation of the BCLl 1A gene in these circumstances
results in increased expression of gamma globin, and the number of off-target
cleavage events is reduced.
In some aspects, the ion comprises a non-naturally occurring
zinc finger protein comprising a zinc finger protein (ZFP) comprising 4, 5 or 6
fingers, each finger comprising a recognition helix region that recognizes a DNA
target subsite n the recognition helix s comprise the sequences in the
order shown in a single row of Table 1. Within each zinc finger, the 7 amino acid
recognition helix region is numbered -1 to +6 within the zinc finger backbone (of
approximately 30 residues, including zinc coordinating residues). In certain
embodiments, 1, 2, 3 or more of the component zinc fingers of the zinc finger proteins
described herein further se mutations to one or more residues outside the
recognition helix region, including but not limited to mutations to amino acids at
position -5, position -14 or at both positions -5 and -14 ring continuing from
the -1 to +6 numbering used for the recognition helix region) are mutated. See, e. g.,
Qm4 and le4 mutations described in US. Provisional Patent Applications
62/378,978 and 62/443,981. The component zinc fingers of the zinc finger protein
can be linked by any linkers, for example as described in US. Patent No. 8,772,453.
In certain embodiments, the ZFP ses the recognition helixes as shown in Table
1 for the proteins designated as follows: 63014 (which binds to the target site shown
in SEQ ID NO: 1) and 65722 (which binds to the target site shown in SEQ ID N02).
In certain ments, the zinc finger proteins as bed herein are
2O fused to a functional domain (e.g., transcriptional activation domain, transcriptional
repression domain, cleavage domain (to form a zinc finger nuclease), etc.). Any
linker may be used to operably link the cleavage domain and the zinc finger protein,
including but not limited to s as described in US. Patent Nos. 9,394,531 and
9,567,609. Furthermore, when a FokI cleavage domain is used, further mutations in
the catalytic domain, dimerization domain, to phosphate contact residues (not in the
zation or catalytic ), and ations of ons in any one of the
catalytic domain, dimerization domain and to phosphate contact residues may be
present, including but not limited to ELD or KKR mutations to the dimerization
domain, mutations to residues 525 (K to S) of the FokI domain, and combinations of
ELD or KKR mutations to the dimerization domain and mutations to residues 525 (K
to S) of the FokI domain, numbered relative to wild-type. See, US. Patent Nos.
7,888,121, 7,914,796, 8,034,598, 618 and US. Patent Publication No.
2011/0201055 and US. Provisional Patent Application Nos. 62/378,978 and
62/443 ,98 l .
In certain embodiments, zinc finger nucleases (ZFNs) may be used in
dimerizing pairs to cleave at or near one or both of the target sites for the ZFNs of the
pair, for example, the “left partner” of Table l (e.g., 63014) can form a dimer with the
“right partner” of Table l (e. g., 65722) to cleave BCLl 1A enhancer sequences. In
certain ments, the pair of ZFNs comprises the following amino acid
sequences:
63 0 l 4:
MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAMAERPFQCRI
CMQNFSDQSNLRAHIRTHTGEKPFACDICGRKFARNFSLTMHTKIHTGSQKPF
QCRICMQNFSSTGNLTNHIRTHTGEKPFACDICGRKFATSGSLTRHTKIHTHPR
APIPKPFQCRICMQNFSDQSNLRAHIRTHTGEKPFACDICGRKFAAQCCLFHHT
KIH— Linker -
ELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGK
HLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMERYVEENQ
TRDKHLNPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGA
VLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS (SEQ ID N023), and
MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAMAERPFQCRI
CMQKFARNDHRTTHTKIHTGEKPFQCRICMQNFSQKAHLIRHIRTHTGEKPFA
CDICGRKFAQKGTLGEHTKIHTGSQKPFQCRICMQNFSRGRDLSRHIRTHTGE
KPFACDICGRKFARRDNLHSHTKIH— Linker-
ELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGK
HLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVKENQ
TRNKHINPNEWWKVYPSSVTEFKFLFVSGHFSGNYKAQLTRLNRKINCNGA
LLIGGEMIKAGTLTLEEVRRKFNNGEINF (SEQ ID NO:4), wherein the
Linker ce can be any linker sequence known in the art, for e as
described in US. Patent Nos. 9,394,531 and 9,567,609. In certain embodiments, the
linker for 63014 comprises or consists of the L7c5 linker SRARPLNPHP
(SEQ ID N05» and the linker used in 65722 comprises or consists of the L0 linker
(LRGSQLVKS (SEQ ID NO:6), see US. Patent No. 9,567,609). The FokI cleavage
domain sequence C-terminal to the Linker of the sequences shown above may also
comprise alternative FokI domains operably linked to the zinc finger protein. In
certain embodiments, the FokI cleavage domain may include alternate or additional
ons to the catalytic domain, the dimerization domain, phosphate contact
es, and combinations of mutations to any one of the catalytic domain,
dimerization domain and phosphate t residues.
In another aspect, the invention comprises delivery of at least one
se (e.g., a nuclease that binds to a BCLl 1A er sequence) to a human
stem cell or precursor cell (HSC/PC) for the purpose of genome engineering. In
n embodiments, the nuclease comprises a zinc finger protein (ZFP) comprising
4, 5 or 6 fingers, each finger comprising a recognition helix region that recognizes a
target subsite wherein the recognition helix regions comprise the sequences in the
order shown in a single row of Table 1. In other embodiments, the ZFN nuclease
comprises the pair of ses designated 65722. 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 609,
including but not limited to LRGSISRARPLNPHP (SEQ ID N05) or (LRGSQLVKS
(SEQ ID NO:6)).
[0022] 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 red ments, 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 d (See e.g.
Kormann el al. (201 1) Nature hnology 29(2): 154-157). In other aspects, the
mRNA may comprise an ARCA cap (see US. Patent Nos. 7,074,596 and 8,153,773).
In further embodiments, the mRNA may comprise a mixture of unmodified and
modified nucleotides (see US. Patent Publication No. 2012/0195936). 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 a transcription factor. In some aspects, the transcription factor is GATA-l.
In other aspects, the invention comprises a cell or cell line in which an
endogenous BCLl 1A enhancer sequence is genetically modified by a se as
described herein (e.g., shown in Table l), for example as compared to the wild-type
sequence of the cell. The genetic modification to the BCLl lA enhancer s in
modification of globin (beta and gamma) gene expression. Nuclease-modified cells
or cell lines as described herein are distinguishable in structure, function, and
combinations of both structure and function from wild-type. The genetically modified
cell or cell lines may be heterozygous or homozygous for the modification. The
modifications may comprise insertions (e.g., transgene ion) ons, and
combinations of insertions and deletions; such insertions, deletions, and combinations
of insertions and deletions are commonly referred to as “indels”. In some preferred
embodiments, indels result in the destruction of a transcription factor binding site. In
certain embodiments, the modification is at or near the nuclease(s) binding site(s),
cleavage ), and combinations of binding and cleavage sites, for example, within
1-300 (or any value etween) base pairs upstream or downstream of the ) of
cleavage, more preferably within l-lOO base pairs (or any value therebetween) of
either side of the binding site(s), cleavage site(s), and combinations of binding and
cleavage sites shown in Table 1, even more preferably within 1 to 50 base pairs (or
any value therebetween) on either side of the binding site(s), cleavage site(s), and
combinations of g and cleavage sites. In n embodiments, the genetic
modification of the BCLl lA enhancer ce is within and/or between ces
shown in Table 1 (target sites). The modification may also include modifications to
one or more nucleotides in the cleavage sites. The modification may also include
modifications to one or more nucleotides in the binding sites. The ation may
further include modifications to one or more nucleotides in the cleavage sites, and in
one or more of the binding sites. In certain embodiments, one or more of the nuclease
target site(s) is(are) not modified. In other embodiments, at least one of the target
sites for the nuclease(s) is(are) modified. In certain embodiments, the modification is
at or near the “+58” region of the BCLl lA enhancer, for example, at or near a
nuclease binding site shown in any of SEQ ID N01 and SEQ ID NO:2. Any cell or
cell line may be modified by the nucleases as described herein, for example a stem
cell (hematopoietic stem cell such as a CD34+ hematopoietic stem cell) or red blood
cell (RBC) precursor cell.
Also bed are cells or cell lines obtained ing modification
by a nuclease as described herein, for example cells or cell lines descended from a
nuclease-modified cell or cell line as described herein. Partially or fully differentiated
cells ded from the modified stem cells as described herein are also provided
(e.g., RBCs or RBC precursor cells). The cells descended from the nuclease-modified
cells may be propagated, differentiated, and combinations of both propagated and
differentiated in vitro (culture) or may differentiate within a live subject, for example
following ex vivo administration of a nuclease-modified stem cell. Any of the
genetically modified cells or cell lines sed herein may show increased
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expression of gamma globin. Compositions such as pharmaceutical compositions
comprising the genetically modified cells as described herein are also provided.
In other s, the invention comprises delivery of a donor nucleic
acid to a target cell to provide a genetically modified cell in which the donor is
ated into the cell. The donor may be delivered prior to, after, or along with the
nucleic acid encoding the nuclease(s) of Table l. The donor nucleic acid may
comprise an exogenous sequence (transgene) to be integrated into the genome of the
cell, for e, an endogenous locus. In some embodiments, the donor may
comprise a full-length gene or nt thereof flanked by regions of homology with
the ed ge site. In some embodiments, the donor lacks homologous regions
and is integrated into a target locus through homology independent mechanism (1’. 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., BCLl 1A 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 tion event,
or a targeted deletion.
[0026] In other aspects, the nuclease, donor, and combinations of both the
nuclease and donor is(are) red by viral, non-viral, and combinations of viral and
non-viral gene er methods. In red 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 pe in comparison with the capsid serotype.
[0027] In some aspects, deletions comprising regions within the DNAseI
hypersensitive regions of the enhancer (e.g., the +58 region of the BCLl 1A enhancer)
are made using one or more nucleases as shown in Table 1. These deletions can
comprise from about 1 tide 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-l binding site, or the binding site for
GATA-l in combination with other factors.
In some embodiments, the DNA binding domains of Table l 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 ments,
the fusion proteins comprise the DNA binding domain of Table l fused to a gene
expression modulatory domain where the tor represses gene expression.
In some embodiments, the HSC/PC cells are ted with the
ses, the DNA binding proteins, and combinations of the nucleases and DNA
binding proteins of the invention (i.e., ZFPs as shown in Table 1). In some
embodiments, the nucleases, the DNA binding proteins, and ations of the
nucleases and 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, the DNA binding proteins, and combinations of the
ses and DNA binding proteins, and in further embodiments, the mRNAs may
be protected. In some embodiments, the mRNA may be chemically modified, may
se an ARCA cap, a mixture of unmodified and modified nucleotides, and
combinations of an ARCA cap and a mixture of unmodified and modified
nucleotides. Cells or cell lines descended from these cells are also provided,
including lly or fully entiated cells.
In some aspects, the HSC/PC are contacted with the nucleases, the
DNA binding proteins, and combinations of the nucleases and DNA binding proteins
of the invention ex vivo, following apheresis of the HSC/PC from a subject, or
purification from harvested bone marrow. In some embodiments, the ses
described herein cause modifications within the BCLl lA enhancer regions, for
example resulting a cally modified cell that is structurally, functionally, and
combinations of structurally and functionally ct from wild-type cells, other
modified (e.g., nuclease-modified) cells, and combinations of wild-type and other
modified cells. In further embodiments, the HSC/PC containing the BCLl lA
enhancer region modifications are introduced back into the subject. In some
instances, the HSC/PC containing the BCLl lA enhancer region modifications are
expanded prior to introduction. In other aspects, the genetically modified HSC/PCs
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 ed from
the subject following G—CSF-induced mobilization, afor-induced mobilization,
and combinations of G—C SF- and plerixafor-induced mobilization, and in others, the
cells are isolated from human bone marrow or human umbilical cords. In some
s, the subject is d to a mild myeloablative procedure prior to introduction
of the graft comprising the modified HSC/PC, while in other s, the subject is
d with a vigorous myeloablative conditioning n. 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 thalassemia. In some
aspects, the hemoglobinopathy is a beta semia, while in other aspects, the
hemoglobinopathy is sickle cell disease.
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 onal
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, ing but not limited to, sequences
encoding a 2A peptide, SA site, IRES, etc.
In one aspect, the methods and itions of the invention comprise
methods for contacting the HSC/PC in vivo. The nucleases, DNA binding proteins, or
ation of nucleases and DNA binding proteins are red to HSC/PC in silu
by methods known in the art. In some embodiments, the nucleases and/or DNA
binding proteins of the invention comprise a viral le that is administered to the
subject in need, while in others, the nucleases, DNA binding proteins, or combination
of nucleases and DNA binding proteins comprise a nanoparticle (e.g. liposome). In
some embodiments, the viral particles, nanoparticles, or combination of viral particles
and nanoparticles are delivered to the organ (e.g. bone marrow) wherein the HSC/PC
reside.
In another aspect, bed 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 as bed herein, 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 ce may be cleaved by one
or more of the same ses used to cleave the endogenous gene into which
integration is desired. In certain embodiments, the donor sequence includes different
nuclease target sites from the ses 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 that is ered to bind a sequence within
the region of interest, and a cleavage domain or a cleavage half-domain.
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, modulates expression
of, or both binds to and modulates expression of a gene of interest. In one
embodiment, the regulatory proteins bind to a DNA sequence and prevents binding of
other regulatory s. In another embodiment, the binding of the regulatory n
may modulate (i.e. induce or repress) expression of a target DNA.
[0035] In some embodiments, the transgenic HSC/PC cell, transgenic animal,
or combination of enic HSC/PC cell and animal includes a transgene that
encodes a human gene. In some instances, the transgenic animal comprises a knock
out at the endogenous locus, and ement of the endogenous gene with its human
counterpart, thereby allowing the pment of an in vivo system where the human
protein may be studied in isolation. Such transgenic models may be used for ing
purposes to identify small molecules or large biomolecules or other entities which
may ct with or modify the human protein of interest. In some aspects, the
transgene is integrated into the ed 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.
In another aspect, provided herein is a method of altering gene
expression (e.g., BCLl 1A, a globin gene, and combinations of BCLl 1A and a globin
gene) in a cell, the method comprising: introducing, into the cell, one or more
nucleases as described herein (shown in Table 1), under ions 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
ating a donor at or near the site of nuclease ge in the BCLl lA gene. The
donor sequence is introduced to the cell using a viral vector, as an oligonucleotide, on
a plasmid, and combinations of one or more s selected from using a viral
vector, as an oligonucleotide, or on a plasmid. The cell in which gene expression is
altered may be, for example, a red blood cell (RBC) precursor cell, a hematopoietic
stem cell (e.g., CD34+ cell), and ations of RBC precursor cell and a
hematopoietic stem cell.
In other embodiments, provided herein is a method of producing a
genetically d cell comprising a c modification within an endogenous
BCLl lA enhancer sequence (a ation to the nucleotide sequence of the
BCLl lA 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 l, b)
subjecting the cell to conditions conducive to expressing the zinc finger protein from
the polynucleotide, and c) modifying the endogenous BCLl lA enhancer ce
with the expressed zinc finger n sufficient to produce the genetically modified
cell. In certain embodiments, the cells are ated 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).
Cells comprising one or a combination of the c modifications
bed herein are also provided, including cells descended from the cells produced
by the methods described herein.
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, wherein the pharmaceutical preparation comprises
genetically modified cells, proteins, cleotides, and ations of one or
more selected from genetically modified cells, proteins, and polynucleotides, as
described herein in an amount sufficient to increase the globin gene expression in the
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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 e.
A kit, comprising the c acids, proteins, genetically modified
cells, and combinations of one or more selected from the c acids, proteins, and
genetically modified cells of the invention, is also provided. The kit may comprise
nucleic acids encoding the ses, (e.g. RNA molecules or ZFN, TALEN or
CRISPR/Cas system encoding genes contained in a suitable expression vector),
aliquots of the nuclease proteins, donor molecules, suitable modifiers of stem cell
enewal (“stemness”), cells, buffers, instructions (e.g., for performing the
methods of the invention) and the like, including various combinations of these kit
components. The invention includes, but is not limited to, a genetically modified cell
(e.g., stem cell such as a hematopoietic (CD34+) stem cell or RBC sor cell)
comprising at least one genomic modification made by a nuclease (e.g., as shown in a
single row of Table 1), wherein the genomic modification is within an endogenous
BCLl lA er sequence, and further wherein the genomic modification is
selected from the group consisting of insertions, deletions and ations thereof
and comprises a modification at, near or between any of SEQ ID N01 and SEQ ID
NO:2. In certain embodiments, the cell is a genetically modified differentiated cell
descended from a stem cell as described herein (e.g., a RBC descended from a
poietic stem cell or RBC precursor cell).
The nuclease may comprise at least one zinc finger nuclease (ZFN)
(e.g., as shown in Table l), at least one TALEN, and combinations of at least one
ZFN and at least one TALEN. The nuclease(s) may be introduced into the cell in
protein form, as a polynucleotide encoding the nuclease(s), or as a combination of
protein form and cleotide encoding the nuclease(s). In certain embodiments,
the genomic modification comprises an insertion that comprises integration of a donor
polynucleotide encoding a transgene. Also provided are pharmaceutical compositions
comprising one or more of the genetically modified cells as described herein.
Also provided is a DNA-binding protein comprising a zinc finger
protein comprising 4, 5 or 6 zinc finger domains comprising a ition helix
region, wherein the zinc finger proteins comprise the recognition helix regions in the
order shown in a single row of Table 1. Also provided is a TALE n comprising
a ity of repeats that bind to a sequence comprising a portion (e.g., at least 4, 5, 6
or more) base pairs of the target sites shown in Table l. A fusion protein comprising
a zinc finger n or TALE protein as bed herein and a wild-type or
engineered cleavage domain or cleavage half-domain is also provided as are
polynucleotides encoding the proteins (ZFPs, TALEs, ZFNs, TALENs) as described
herein. Cells (e.g., isolated stem cells such as hematopoietic (CD34+) stem cells)
comprising one or more polynucleotides, proteins, and combinations of
polynucleotides and proteins as bed herein are also provided. Also provided are
kits comprising one or more ns, polynucleotides, cells, or combinations f
as described herein.
A method of altering globin gene expression in a cell (e.g., RBC
sor cell, hematopoietic stem cell and combinations of RBC precursor cell and
hematopoietic stem cell) is also described, the method comprising: introducing, into
the cell, one or more polynucleotides encoding one or more nucleases as described
herein, under conditions such that the one or more proteins are expressed and
expression of the globin gene (e.g., gamma globin, beta globin, and combinations of
gamma and beta globin) is altered (e.g., increased). In certain embodiments, the
methods further comprise integrating a donor sequence into the genome of the cell,
for e using a viral vector, as an oligonucleotide or on a plasmid. The donor
sequence may comprise a transgene under the control of an endogenous or exogenous
[0044] Also provided is a method of producing a genetically modified cell
sing a genomic modification within an endogenous BCLl lA enhancer
sequence (e.g., target site as shown in Table l), the method comprising the steps of:
(a) ting 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 l, (b) subjecting the cell to conditions conducive to expressing
the fusion protein from the polynucleotide, and (c) modifying the endogenous
BCLl lA enhancer sequence with the expressed fusion protein sufficient to produce
the genetically modified cell. In certain ments, the method further comprises
stimulating the cells with at least one cytokine. The cleotide(s) may be
delivered inside the cell, for example using a non-viral delivery system, a viral
delivery system, a delivery vehicle, and combinations selected from a non-viral
delivery system, a viral ry system, and a delivery vehicle and may comprise
subjecting the cells to an electric field or employing cell membrane disruption as a
delivery mechanism (so called ‘Squeeze Technology’, see e.g. Sharei el al. (2015)
PLOS ONE doi: l/journal/pone.Ol18803).
Methods of treating a patient in need of an se in globin gene
expression (e.g., a patient is known to have, is suspected of having, or is at risk of
developing a hemoglobinopathy such as a semia (e.g., B-thalassemia) or sickle
cell disease are also provided, the method comprising administering to the patient the
pharmaceutical composition as described herein (e.g., proteins, polynucleotides, cells
or a combination selected from proteins, polynucleotides and cells) in an amount
sufficient to increase the globin gene expression in the patient.
[0046] These and other aspects will be readily apparent to the skilled n in
light of disclosure as a whole.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A and 1B are schematics depicting an ew of the
oligonucleotide duplex integration site assay. Figure 1 shows the first step of the
assay, in which cells are treated with ZFNs in the presence of supplemental
oligonucleotide duplex DNA, which is captured into a fraction of the resulting
cleavage events. Figure 1B shows the subsequence step in which cells are cultured
for seven days, genomic DNA is isolated, and segments of the genome flanking donor
ation sites are amplified via adaptor-mediated PCR using a primer to the
integrated oligonucleotide duplex. Amplicons are then sequenced to reveal ate
cleavage sites.
Figure 2 depicts the location of 455 potential off-target loci identified
by the oligonucleotide duplex integration site assay using the 51949 original or
parent ZFN pair. The top 63 loci are highlighted in gray and were analyzed in follow
up indel analyses. Each locus is identified by the chromosome and base number
indicating the most likely on of cleavage, as well as an indication of the number
of integrants detected at that locus.
Figures 3A through 3C depict exemplary phosphate contacting
residues within the zinc finger scaffold. Figure 3A depicts a zinc finger interaction
with a DNA molecule, and shows the location of the wild type arginine side chain and
how it interacts with the phosphate backbone of the DNA molecule. Figure 3B (SEQ
ID Nos:7 to 17) depicts exemplary ZFN sequences showing where this ne is
located in the primary sequence of each zinc finger (vertical row of ‘R’ residues
WO 39440
indicated via bold arrow) and also ghts those arginines that were substituted
with glutamine in the ZFP backbone to eliminate the corresponding phosphate contact
idually boxed ‘R’ residues). The sequences also show the ition helix
regions (where residues -1 to +3, +5 and +6 are boxed and ) as well as a
portion of the linker between the C-terminal zinc finger domain and the cleavage
domain (cleavage domain not shown). Figure 3C further depicts the spatial location
of another potential backbone contacting lysine sidechain present in the FokI cleavage
domain, which during specificity optimization may be substituted to a serine.
Figure 4 is a graph comparing ation levels via treatment of
CD34 cells with either the original 51857/51949 pair or with the optimized
63014/65722 pair at various tested loci. Shown across the bottom or on the ,
are the loci identified as potential cleavage targets, with the percent indels for each
site shown on the al or y-axis. Note that the y-axis is shown in a log scale. The
dark gray bars show the loci cleaved by the 51857/51949 pair, and the amount of
cleavage detected, while the light gray bars are those loci cleaved by the optimized
pair where nearly all cleavage measured is at the targeted BCLl la target sequence.
Figures 5A and 5B are graphs depicting the relative ratios of globin
mRNAs made in hCD34+ cells following treatment with BCLl 1A specific ZFNs and
erythroid entiation. CD34+ cells derived from two healthy human donors (PB-
MR-OO3 or PB-MR-OO4) were treated or not treated with the ZFN pair and then 0L, [3,
and v globin expression was ed. The best method to determine the amount of v
globin mRNA found following ZFN treatment is to express the change in expression
as either a ratio of v globin to B globin (Figure 5A), or y globin to 0t globin mRNA
(Figure 5B).
[0052] Figure 6 is a graph depicting the relative s of v-globin protein
produced in the treated CD34+ cells. As above, two CD34+ cell lots derived from
healthy human donors were used (PB-MR-OO3 and PB-MR-OO4). In this experiment,
an approximate 3-4 fold elevation of fetal globin protein percentages to levels of
about 15%-20% was observed in erythroid progeny of HSPCs upon 63014/65722-
ed disruption of the BCLl 1A enhancer in both donor lots.
Figure 7 is a graph indicating relative human chimerism in mice
engrafted with 63014/65722 treated donor lots (“+ZFN”) as described above. Human
chimerism was measured through the detection of cells bearing a hCD45 marker on
their surface using FACS. Percentages of human hCD45+ cells in the peripheral
WO 39440 2017/048397
blood collected at either 8 or 12 weeks post-transplant are indicated. The data showed
good engraftment levels in this study with comparable human chimerism following
engraftment of untransfected control (“(-)”) and ZFN transfected HSPC (“+ZFN”).
Open circles and triangles represent individual animals.
Figure 8 shows the percentage of chimerism detected in the bone
marrow of the engrafted mice where human cells were identified by the presence of
hCD45 on their cell surfaces. Samples were analyzed at 12 weeks post-engraftment.
Figures 9A through 9D are graphs depicting the titution of
various hematopoietic cell lineages tested by FACS analysis of bone marrow cells in
engrafted mice obtained at week 12 with antibodies recognizing lineage specific cell
surface markers. The data showed comparable entation of all analyzed human
hematopoietic lineages in the bone marrow at week 12 post-injection between the
BCL11A-specific ZFN mRNA treated CD34+ cell progeny (“ ‘14/’22 “) and that of
the untransfected cells (“(-)”). Shown are data from id, myeloid, erythroid and
HSPC (Figures 9A through 9D, respectively) for cells derived from both donors
(“003” and “004”). The data showed comparable representation of all analyzed
human hematopoietic lineages in the bone marrow at week 12 post-injection between
the Bcll 1A ZFN mRNA treated CD34+ cell progeny and that of the untransfected
cells.
2O [0056] Figure 10 is a graph ing the level of gene modification at the
BCL11A target in DNA isolated from the peripheral blood of engrafted mice, assayed
by deep sequencing. Data are shown for the input cells (2 days after ZFN
transfection, (“+”)), and then for blood cells 8 or 12 weeks following engraftment, and
demonstrated a good retention of gene modification. Untransfected cells are
ented by “(-)” in the Treatment line.
Figure 11 is a graph ing the amount of gene modification at the
BCL11A target for bone marrow cell samples following engraftment of the ZFN-
treated cells (“+”). ted cells are represented by “(-)” in the Treatment line.
Comparable modification was observed in both BCL11A dependent lineages (B cells,
expressing the CD19 marker, primitive progenitors, expressing CD45 and high levels
of CD38) and BCL11A independent (myeloid) lineages. gh the input gene
modification levels were higher in the PB-MR-OO3 donor sample than in the PB-MR-
004 donor sample, the PB-MR—OO4 d cells consistently showed higher
modification levels, 1'.e. better retention of modification, in mice than those derived
from OO3.
Figure 12 is a graph depicting the amount of gene modification in
erythroid cells derived from week 12 bone marrow cells following in vitro
differentiation for 14 days. The data were from mice originally engrafted with the
two different donors described above, and trated that the BCLl 1A
modification ed by ZFN treatment (“+ ZFN”) is not markedly altered during
the erythroid differentiation. Cells that were not treated with ZFN are indicated by
“(-) ZFN”.
[0059] Figures 13A and 13B are graphs depicting the relative amount of
y-globin encoding mRNA, where the concentration of y-globin mRNA is depicted
either as a ratio of y /B globin mRNA (Figure 9A) or as a ratio of y globin /0L
globin mRNA (Figure 9B). Both in the untransfected (“(-) ZFN”) and the ZFN
treated samples (“+ ZFN”) y-globin to B globin or in to OL- globin mRNA ratios
differed widely n the erythroid progenies of individual mice from the same
group. r, despite this variability and the variability uced by the use of
two different human donors, the 63014/65722 treated sample averages show an ~l .5-2
fold increase in y globin mRNA levels compared to their respective untransfected
counterparts.
2O [0060] Figure 14 is a graph depicting the difference in the amount of y-globin
protein (expressed as either a ratio of y-globin/d-globin or y-globin/total B-like
n) in bone marrow derived cells from engrafted mice where the bone marrow
cells were submitted to an in vitro differentiation protocol. Protein levels were
measured 16 days into differentiation. The Gamma (y) globin (sum of the Agamma
and Ggamma peaks) to alpha (0L) globin ratios were determined, as well as the Gamma
globin (sum of the Agamma and Ggamma peaks)/ over beta-like globin ratios (sum of
the Agamma, Ggamma, beta and delta- globin peaks). In line with the poor oid
differentiation of the PB-MR—OO3 derived samples the gamma-globin levels in the
untransfected cells derived from this donor were very high (~3 0%), and therefore
ZFN treatment (“+ ZFN”) resulted in only a 12-fold increase in gamma-globin levels
as compared with untreated cells (“(-) ZFN”). The PB-MR—OO4 showed more typical
untransfected levels (~9%) and exhibited an ~2-fold increase in gamma-globin protein
levels after 12 weeks passage through the mouse.
DETAILED DESCRIPTION
Disclosed herein are itions and methods for genome
engineering for the modulation of BCLl lA, gamma globin, and combinations of
BLCl 1A and gamma globin expression and for the treatment, prevention, or
treatment and tion of hemoglobinopathies. In particular, via targeting with
nucleases comprising the ZFPs having the recognition helix regions as shown in a
single row of Table l, disruption of an enhancer of BCLl 1A is efficiently achieved in
HSC/PC and s in a change in relative gamma globin expression during
sub sequent erythropoiesis. This modulation of BCLl 1A and gamma globin
IO 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 d sequelae caused by the
aberrant beta globin can be overcome by alteration of the expression of gamma globin
in erythrocyte precursor cells.
General
Practice of the methods, as well as preparation and use of the
compositions disclosed herein employ, unless ise indicated, conventional
techniques in molecular biology, biochemistry, chromatin structure and analysis,
computational try, cell culture, inant DNA and related fields as are
within the skill of the art. These techniques are fully ned in the ture. See,
for example, Sambrook el al. MOLECULAR CLONING: A LABORATORY
MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third
n, 2001, Ausubel el 61]., CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY, John Wiley & Sons, New York, 1987 and ic updates, the series
METHODS IN ENZYMOLOGY, Academic Press, San Diego, Wolffe,
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
S IN MOLECULAR BIOLOGY, Vol. 119, “Chromatin Protocols” (PB.
Becker, ed.) Humana Press, Totowa, 1999.
Definitions
The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” are
used hangeably and refer to a deoxyribonucleotide or ribonucleotide polymer, in
linear or ar 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
l nucleotides, as well as nucleotides that are modified in the base, sugar,
phosphate moieties (e.g., phosphorothioate backbones), and combinations selected
from base, sugar and phosphate moieties. In general, an analogue of a particular
nucleotide has the same base-pairing specificity, 1'. e., an analogue of A will base-pair
with T.
The terms eptide,” “peptide” and “protein” are used
interchangeably to refer to a polymer of amino acid es. 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.
“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 (Kd) of 10'6 M'1 or lower. “Affinity” refers to the th of binding:
sed binding affinity being correlated with a lower Kd.
A “binding protein” is a n that is able to bind to r
molecule. A binding protein can bind to, for example, a DNA molecule (a
DNA-binding protein), an RNA molecule (an RNA-binding protein), a protein
le (a protein-binding protein), or can bind to a combination of molecules
ed from a DNA molecule, an RNA molecule or a protein. In the case of a
protein-binding protein, it can bind to itself (to form homodimers, homotrimers, etc),
it can bind to one or more molecules of a different protein or ns, or it can bind to
both itself and one or 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.
A “zinc finger DNA binding protein” (or binding ) is a protein,
or a domain within a larger protein, that binds DNA in a sequence-specific manner
through one or more zinc , 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.
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 t 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.
[0069] Zinc finger and TALE binding domains can be “engineered” to bind to
a predetermined nucleotide sequence, for example via ering (altering one or
more amino acids) of the ition 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 ion. A designed
DNA binding protein is a protein not occurring in nature whose design/composition
s principally from rational criteria. Rational criteria for design include
application of tution rules and computerized thms for processing
information in a database storing ation of existing ZFP and/or TALE designs
and binding data. See, for example, US. Patent Nos. 6,140,081, 6,453,242, 6,534,261
and 8,585,526, see also PCT Publication Nos. WO 98/53058, WO 98/53059,
WO 98/53060, WO 536 and WO 03/016496.
A “selected” zinc finger protein or TALE is a protein not found in
nature whose production results primarily from an cal process such as phage
display, interaction trap or hybrid selection. See e.g., US. Patent Nos. 5,789,538,
523, 6,007,988, 6,013,453, 6,200,759, 526, PCT Publication Nos.
WO 95/19431, WO 96/06166, WO 98/53057, WO 98/54311, WO 00/27878,
WO 01/60970 WO 01/88197, WO 02/099084.
“TtAgo” is a prokaryotic ute protein thought to be involved in
gene silencing. TtAgo is derived from the bacteria Thermus lhermophilus. See, e.g.,
Swarts el al. ibid, G. Sheng el al. (2013) Proc. Natl. Acad. Sci. USA. 111, 652). A
“TtAgo system” is all the components required including, for example, guide DNAs
for cleavage by a TtAgo enzyme.
“Recombination” refers to a process of exchange of genetic
information n two polynucleotides, including but not limited to, donor capture
by non-homologous end g (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 s
requires nucleotide sequence homology, uses a “donor” molecule to template repair of
a “target” molecule (1'.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 c 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, esis-dependent strand ing,” in which the donor is
used to resynthesize genetic ation that will become part of the target, d
processes, or combinations thereof. 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 cleotide.
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 indels by
homology-directed repair or by non-homology-directed repair mechanisms. Deletions
may include any number of base pairs. Similarly, ions 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 tide sequence as
in the donor into the ar chromatin. Thus, a first sequence in cellular chromatin
can be altered and, in n 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, (1'.e.,
replacement of a ce in the informational sense), and does not necessarily
require physical or chemical replacement of one polynucleotide by another.
In any of the s described herein, additional pairs of zinc-finger
proteins or TALEN can be used for additional double-stranded cleavage of onal
target sites within the cell.
Any of the methods described herein can be used for insertion of a
donor of any size, 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.
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
ce 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-l,OOO base pairs (or any integral value etween) or any number
of base pairs greater than 1,000, that are gous or cal 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.
“Cleavage” refers to the breakage of the nt backbone of a DNA
molecule. Cleavage can be initiated by a variety of methods including, but not limited
to, tic or chemical hydrolysis of a phosphodiester bond. Both single-stranded
cleavage and double-stranded ge are le, 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 ptides are used for ed -stranded DNA
cleavage.
A “cleavage half-domain” is a polypeptide sequence which, in
conjunction with a second ptide (either cal or different) forms a complex
having ge activity (preferably double-strand cleavage activity). The terms “first
and second cleavage half-domains,” “+ and — cleavage half-domains” and “right and
left cleavage omains” are used interchangeably to refer to pairs of cleavage half-
s that dimerize.
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, US. Patent Nos.
7,888,121, 7,914,796, 8,034,598, 8,623,618 and US. Patent Publication No.
2011/0201055, incorporated herein by reference in their entireties.
The term “sequence” refers to a tide 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 r etween), more preferably between about 2000 and
,000 nucleotides in length (or any value etween) and even more preferable,
2O n about 5 and 15 kb (or any value therebetween).
“Chromatin” is the nucleoprotein structure comprising the cellular
genome. Cellular chromatin comprises nucleic acid, primarily DNA, and protein,
including es and non-histone chromosomal ns. The majority of
eukaryotic cellular chromatin exists in the form of nucleosomes, wherein a
nucleosome core comprises approximately 150 base pairs ofDNA 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 t disclosure, the term “chromatin” is meant to encompass all types of
cellular nucleoprotein, both prokaryotic and eukaryotic. Cellular chromatin includes
both somal and episomal chromatin.
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.
An “episome” is a replicating nucleic acid, nucleoprotein complex or
other structure comprising a c acid that is not part of the chromosomal
karyotype of a cell. Examples of episomes include plasmids and certain viral
genomes.
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
ed that an accessible region is one that is not ed into a nucleosomal
structure. The distinct structure of an accessible region can often be detected by its
sensitivity to chemical and enzymatic , for example, nucleases.
A “target site” or “target sequence” is a nucleic acid sequence that
defines a n of a nucleic acid to which a binding molecule will bind, provided
sufficient conditions for binding exist.
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. l presence in the cell” is determined with respect to the particular
developmental stage and environmental conditions of the cell. Thus, for e, a
le that is present only during embryonic development of muscle is an
exogenous le 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 nous molecule.
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,
ccharide, any modif1ed derivative of the above molecules, or any complex
sing one or more of the above les. 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, US. Patent Nos. 5,176,996 and
,422,251. Proteins e, but are not limited to, DNA-binding proteins,
transcription factors, chromatin remodeling factors, methylated DNA binding
proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases,
phosphatases, ases, recombinases, ligases, topoisomerases, gyrases and
helicases.
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
ous nucleic acid can se 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 (1'. e.,
liposomes, including neutral and cationic lipids), electroporation, direct ion, cell
fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-
mediated er 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 ce
may be introduced into a cell line originally derived from a mouse or hamster.
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 c acid can comprise a chromosome,
the genome of a mitochondrion, chloroplast or other organelle, or a naturally-
ing episomal nucleic acid. Additional endogenous molecules can include
proteins, for example, transcription factors and enzymes.
As used , the term “product of an exogenous nucleic acid”
includes both polynucleotide and polypeptide ts, for example, transcription
products (polynucleotides such as RNA) and ation products eptides).
A “fusion” molecule is a molecule in which two or more subunit
molecules are linked, preferably covalently. The subunit les 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 d to, fusion
proteins (for example, a fusion between a ZFP or TALE DNA-binding domain and
one or more tion domains) and fusion nucleic acids (for example, a nucleic acid
encoding the fusion n described supra). Examples of the second type of fusion
molecule include, but are not limited to, a fusion between a x-forming nucleic
acid and a polypeptide, and a fusion between a minor groove binder and a nucleic
acid.
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 te the fusion protein. Trans-splicing, polypeptide ge and
ptide ligation can also be involved in expression of a protein in a cell. Methods
for polynucleotide and ptide delivery to cells are presented elsewhere in this
disclosure.
[0093] A “gene,” for the purposes of the t 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 sequences, transcribed sequences, and combinations of coding and
transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to,
promoter ces, terminators, translational regulatory sequences such as me
binding sites and internal ribosome entry sites, enhancers, silencers, tors,
boundary elements, replication origins, matrix attachment sites and locus control
“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,
microRNA, 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, ation, and editing,
and proteins modified by, for example, methylation, acetylation, phosphorylation,
ubiquitination, ADP-ribosylation, myristilation, and ylation.
ation” of gene expression refers to a change in the activity of a
gene. Modulation of expression can include, but is not limited to, gene tion 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 /Cas system as described herein. Thus, gene inactivation may be partial or
complete.
WO 39440 2017/048397
A cted” mRNA is one in which the mRNA has been altered in
some manner to se 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 el al. ((2012), Molecular Therapy, Vol. 16, No. 11, pages
1833 — 1844). Other s include the addition of a so-called ARCA cap, which
increases the translationability of the in vitro produced mRNA (see US. Patent No.
7,074,596).
[0097] A “region of interest” is any region of cellular chromatin, such as, for
example, a gene or a non-coding sequence within or nt to a gene, in which it is
desirable to bind an exogenous le. Binding can be for the purposes of targeted
DNA cleavage, targeted recombination, and ations of targeted DNA cleavage
and targeted recombination. A region of interest can be present in a some, an
episome, an organellar genome (e.g., mitochondrial, chloroplast), or an infecting viral
genome, for e. 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
2O nucleotide pair or up to 2,000 nucleotide pairs in length, or any integral value of
nucleotide pairs.
“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).
The terms “operative linkage” and tively 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 on 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 tory 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 ce 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 uous.
] 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 e, with respect to
a fusion polypeptide in which a ZFP, TALE or Cas DNA-binding domain is fused to
an activation , 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, its binding site, and
combinations of its target site and 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, its binding site, and combinations of its target site and its binding site,
while the cleavage domain is able to cleave DNA in the vicinity of the target site.
A “functional fragment” of a protein, polypeptide or nucleic acid is a
protein, polypeptide or nucleic acid whose sequence is not cal 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 nt can possess more, fewer,
or the same number of residues as the corresponding native le, can contain one
or more amino acid or tide substitutions, and can be combinations possessing
more, fewer, or the same number of residues as the corresponding native molecule
and containing 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 nown. For example, the DNA-binding function of a
ptide can be determined, for e, by fllter-binding, electrophoretic
mobility-shift, or immunoprecipitation assays. DNA cleavage can be assayed by gel
ophoresis. See Ausubel el 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 c and biochemical.
A “vector” is capable of transferring gene sequences to target cells.
Typically, “vector construct,77 (4 expression vector,” and “gene er 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.
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 pigs, cows, rabbits, dogs, cats, rats, mice, and other animals.
Accordingly, the term “subject” or “patient” as used herein means any mammalian
patient or t to which the or stem cells of the invention can be administered.
ts of the present invention e those that have been exposed to one or more
chemical toxins, including, for example, a nerve toxin.
“Stemness” refers to the relative ability of any cell to act in a stem cell-
like , i.e., the degree of toti-, pluri-, multi- or potency and expanded or
nite self-renewal that any ular stem cell may have.
Nucleases
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
lly occurring. In other embodiments, one or more of the nucleases are non-
naturally occurring, i.e., engineered in the DNA-binding domain, the cleavage
domain, and a combination of the DNA-binding domain and cleavage domain. For
example, the nding 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 e binding site). In other embodiments, the nuclease
comprises heterologous DNA-binding and cleavage domains (e.g., zinc finger
ses, TAL-effector domain DNA g proteins, meganuclease DNA-binding
domains with heterologous cleavage domains).
A. DNA-binding domains
In certain embodiments, the DNA binding domain of one or more of
the nucleases used for in vivo cleavage, targeted cleavage of the genome of a cell, and
combinations of in vivo cleavage and targeted cleavage of the genome of a cell
comprises a zinc finger protein. A single zinc finger protein is made up of multiple
WO 39440
zinc finger domains (e.g., 3, 4, 5, 6, or more zinc finger domains). Each zinc finger
domain of is about 30 amino acids in length that it contains a beta turn (containing the
two zinc coordinating residues and an alpha helix (containing the two ant zinc
coordinating residues), which are held in a particular conformation that allow binding
to the protein to a target sequence. Canonical (C2H2) zinc finger domains having two
cysteine (Cys) zinc coordinating residues in the beta turn and two histidine (His) zinc
coordinating residues in the alpha helix or non-canonical (CH3) can be used. See,
e. g., US. Patent No. 9,234,187. A 7-amino acid recognition helix is contained
between the zinc coordinating residues of the beta turn and the zinc coordinating
residues of the alpha helix. The ition helix region is numbered -1 to +6 within
the zinc finger domain and the amino acids outside this recognition region (and
excluding the zinc coordinating residues are referred to as backbone residues).
Preferably, the zinc finger protein is non-naturally occurring in that the
recognition helix is engineered to bind to a target site of choice. See, for example,
See, for example, Beerli el al. (2002) Nature Biotechnol. 20 :135-141 , Pabo el al.
(2001) Ann. Rev. Biochem.70 :313-340 , Isalan el al. (2001) Nature
Biotechnol.19 :656-660 , Segal el al. (2001) Curr. Opin. Biotechnol.12 :632-637 ,
Choo el al. (2000) Curr. Opin. Struct. Biol.10:411-416, US. Patent Nos. 6,453,242,
6,534,261, 692, 6,503,717, 6,689,558, 215; 6,794,136, 7,067,317,
7,262,054, 934; 7,361,635, 7,253,273; and US. Patent Publication Nos.
2005/0064474, 2007/0218528, 2005/0267061, all incorporated herein by reference in
their entireties.
An ered zinc finger binding domain can have a novel binding
specificity, compared to a naturally-occurring zinc finger protein. Engineering
methods include, but are not d to, rational design and s 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 e, co-owned US. Patent Nos. 6,453,242 and 6,534,261,
incorporated by reference herein in their entireties.
rmore, in certain embodiments, the ZFP DNA-binding domains
r comprise on or more modifications to the backbone of one or more the
ent zinc finger domains. The specificity of a ZFP for a target DNA sequence
is dependent upon sequence specific contacts between the zinc finger domains and
specific DNA bases, in particular, between the recognition helix region and the target
site (typically each recognition helix binds to a target subsite of 3 nucleotides). In
addition, the zinc finger domains also comprise amino acid residues that take part in
non-specific interactions with the phosphates of the DNA backbone. Elrod-Erickson
et a]. ((1996) Structure 4:1171) demonstrated through co-crystallization of a zinc
finger protein and its cognate DNA target that there are specific amino acids capable
of interacting with the phosphates on the DNA ne through formation of
hydrogen bonds. Zinc finger proteins that employ the well-known Zif268 backbone
typically have an arginine as the amino terminal e of their second strand of
B-sheet, which is also the second position carboxyl-terminal to the second invariant
cysteine. This position can be referred to as (-5) within each zinc finger domain, as it
is 5th residue ing the start of the d-helix (and position -5 relative to the
recognition helix numbered -1 to +6). The arginine at this position can interact with a
phosphate on the DNA backbone via formation of a charged en bond with its
side-chain guanidinium group. Zinc finger proteins in the Zif268 backbone also
frequently have a lysine at a position that is 4 es amino-terminal to the first
invariant cysteine. This position can be referred to as (-l4) within each finger, as it is
14th residue preceding the start of the d-helix for zinc fingers with two residues
between the zinc coordinating cysteine residues (and position -14 relative to the
recognition helix region numbered -1 to +6). The lysine can interact with a phosphate
on the DNA backbone via formation of a water-mediated d hydrogen bond with
its side-chain amino group. Since phosphate groups are found all along the DNA
backbone, this type of interaction between the zinc finger and a DNA molecule is
generally considered to be non-sequence specific (J. Miller, Massachusetts Institute of
logy Ph.D. Thesis, 2002).
Recent studies have hypothesized that non-specific phosphate
ting side chains in some nucleases may also account for some amount of non-
specificity of those ses (Kleinstiver et a]. (2016) Nature 87):490-5,
Guilinger et a]. (2014) Nut Meth: 429-435). chers have proposed that these
nucleases may possess ‘excess nding energy’, meaning that the nucleases may
have a greater y for their DNA target than is required to substantially bind and
cleave the target site. Thus, attempts were made to decrease the cationic charges in
the TALE DNA binding domain (Guilinger, ibid) or the Cas9 DNA binding domain
(Kleinstiver, ibid) to lower the DNA-binding energy of these nucleases, which
resulted in increased cleavage specificity in vitro. r, additional studies
(Sternberg el al. (2015) Nature 527(7576):l 10-113) also suggest a role in proper
folding and activation of the Cas9 nuclease domain for some of the cationic amino
acids that were mutated in the Kleinstiver study of the Cas9 DNA binding domain.
Thus, the exact role of these amino acids in Cas9 activity is not known.
The methods and compositions of the invention thus include mutations
to amino acids within the ZFP DNA binding domain (‘ZFP backbone’) that can
interact non-specifically with phosphates on the DNA backbone, but they do not
comprise changes in the DNA recognition helices. Thus, the invention includes
mutations of cationic amino acid es in the ZFP backbone that are not required
for nucleotide target specificity. In some embodiments, these mutations in the ZFP
backbone comprise mutating a cationic amino acid residue to a neutral or anionic
amino acid residue. In some embodiments, these ons in the ZFP backbone
comprise mutating a polar amino acid residue to a neutral or lar amino acid
residue. In preferred embodiments, mutations at made at position (-5), on (-9),
position (-l4), and combinations of mutations selected from mutations made at
position (-5), position (-9) and position (-l4) relative to the DNA g helix. In
some embodiments, a zinc finger may comprise one or more mutations at (-5), (-9),
(-l4), and combinations of mutations selected from mutations at (-5), (-9), and (-l4).
In further embodiments, one or more zinc finger(s) in a multi-finger zinc finger
n may comprise ons in (-5), (-9), (-l4) and combinations selected from
(-5), (-9), and (-l4). In some embodiments, the amino acids at (-5), (-9), (-l4) and
combinations ed from (-5), (-9), and (-l4) (e.g. an arginine (R) or lysine (K)) are
d to an alanine (A), leucine (L), Ser (S), Asp (D), Glu (E), Tyr (Y) and/or
glutamine (Q).
In any of these fusion polypeptides described herein, the ZFP partners
may further comprise mutations in the zinc finger DNA binding domain in the (-5),
(-9), (-l4) positions, and ations of mutations selected from mutations at (-5),
(-9), and (-l4). In some embodiments, the Arg (R) at position -5 is changed to a Tyr
(Y), Asp (D), Glu (E), Leu (L), Gln (Q), or Ala (A). In other embodiments, the Arg
(R) at position (-9) is replaced with Ser (S), Asp (D), or Glu (E). In r
ments, the Arg (R) at position (-l4) is replaced with Ser (S) or Gln (Q). In
other embodiments, the fusion polypeptides can comprise mutations in the zinc finger
DNA g domain where the amino acids at the (-5), (-9), (-14) positions, and
ations of mutations selected from ons at (-5), (-9), and (-14) are changed
to any of the above listed amino acids in any combination.
Exemplary selection s, including phage display and two-hybrid
systems, are disclosed in US Patents 5,789,538, 5,925,523, 6,007,988, 453,
6,410,248, 6,140,466, 6,200,759, and 6,242,568, as well as wo 86,
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 e, in co-owned WO 02/077227.
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 US. Patent 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, PCT Publication Nos.
WO 95/19431, WO 96/06166, WO 98/53057, WO 98/54311, WO 00/27878,
WO 70, WO 01/88197, WO 02/099084, WO 58, WO 98/53059,
WO 98/53060, WO 02/016536 and WO 03/016496.
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
2O DNA-binding domains, between the DNA-binding domain and the functional domain
(e.g., nuclease), and between one or more DNA-binding domains and between the
DNA-binding domain and the functional domain. Non-limiting examples of suitable
linker sequences include US. Patent Nos. 8,772,453, 7,888,121, 6,479,626,
6,903,185, and 7,153,949, and US. Patent Publication Nos. 2009/0305419,
2015/0064789 and 2015/0132269. Thus, the proteins described herein may include
any combination of suitable s between the dual DNA-binding components,
between the DNA-binding domain and the functional domain, or between one or more
DNA-binding domains and between the DNA-binding domain and the onal
domain of the compositions described herein.
B. Cleavage Domains
Any suitable cleavage domain can be operatively linked to the DNA-
binding domains as described herein to form a nuclease. 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. Heterologous cleavage domains can
be obtained from any clease or exonuclease. Exemplary endonucleases from
which a ge 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, MA, and Belfort el al. (1997) c Acids Res.
:3379-3388. Additional enzymes which cleave DNA are known (e.g., 81 Nuclease,
mung bean nuclease, pancreatic DNase I, micrococcal nuclease, yeast HO
endonuclease, see also Linn el al. (eds) Nucleases, Cold Spring Harbor Laboratory
Press, 1993). One or more of these enzymes (or onal fragments thereof) can be
used as a source of cleavage domains and cleavage half-domains.
Similarly, a cleavage half-domain can be derived from any nuclease or
n thereof, as set forth above, that es 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 ns are preferably ed, with t to
each other, such that g of the two fusion proteins to their respective target sites
2O 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 zing.
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 tide
pairs or more). In general, the site of cleavage lies between the target sites.
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 s (e.g.,
Type 118) cleave DNA at sites removed from the recognition site and have separable
binding and cleavage domains. For example, the Type 118 enzyme FokI catalyzes
double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one
strand and 13 tides from its ition site on the other. See, for example,
US. Patent Nos. 5,356,802, 5,436,150 and 5,487,994, as well as Li el al. (1992) Proc.
Natl. Acad. Sci. USA 89:4275-4279, Li el al. (1993) Proc. Natl. Acad. Sci. USA
90:2764-2768, Kim el al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887, Kim el al.
) J. Biol. Chem. 269:31,978-31,982. Thus, in one ment, fusion proteins
comprise the ge domain (or cleavage half-domain) from at least one Type 118
restriction enzyme and one or more zinc finger binding domains, which may or may
not be engineered.
For optimal cleavage specificity by a sequence-selective (artificial)
nuclease, it is desirable to arrange conditions so that on-target binding and activity is
not saturating. Under saturating conditions - by definition - an excess of nuclease is
used over what is necessary to achieve complete on-target activity. This excess
provides no on-target benefit but can nonetheless result in increased cleavage at off-
target sites. For monomeric nucleases, saturating conditions may be readily avoided
by performing a simple dose response study to identify and avoid the saturating
plateau on a titration curve. However, for a dimeric se such as ZFN, TALEN
or ok, identifying and avoiding saturating conditions may be more complicated
if the binding affinities of the dual monomers are dissimilar. In such cases, a
dose response study using a simple 1:1 nuclease ratio will only reveal the saturation
point of the weaker g r. Under such a io, if, for example,
monomer affinities differ by a factor of 10, then at the saturation point identified in a
1:1 titration study the higher affinity monomer will be present at a concentration that
2O is 10-fold higher than it needs to be. The resulting excess of the higher affinity
monomer can in turn lead to increased off-target activity without ing any
beneficial increase in cleavage at the intended target, potentially leading to a
decreased specificity overall for any given nuclease pair.
An exemplary Type 118 restriction enzyme, whose cleavage domain is
separable from the binding domain, is FokI. This ular enzyme is active as a
dimer. Bitinaite el al. (1998) Proc. Natl. Acad. Sci. USA 95:10,570-10,575.
Accordingly, for the es of the present disclosure, the portion of the Fok I
enzyme used in the sed fusion proteins is considered a cleavage half-domain.
Thus, for targeted double-stranded cleavage, targeted replacement of cellular
sequences using zinc finger-FokI fusions, and combinations of targeted -
stranded cleavage and targeted replacement of cellular sequences using zinc finger-
FokI fusions, two fusion proteins, each comprising a FokI cleavage half-domain, can
be used to reconstitute a catalytically active cleavage . Alternatively, a single
polypeptide molecule containing a zinc finger binding domain and two Fokl cleavage
half-domains can also be used. ters for targeted cleavage and targeted
sequence alteration using zinc finger-FokI fusions are provided elsewhere in this
disclosure.
A ge domain or cleavage half-domain can be any portion of a
protein that retains cleavage activity, or that s the ability to multimerize (e.g.,
dimerize) to form a functional cleavage domain.
Exemplary Type IIS restriction enzymes are described in US. Patent
No. 7,888,121 incorporated herein in its entirety. Additional restriction s also
contain separable binding and cleavage domains, and these are plated by the
present disclosure. See, for example, Roberts el al. (2003) Nucleic Acids Res. 31:418-
In certain embodiments, the cleavage domain comprises one or more
engineered ge half-domain (also referred to as dimerization domain mutants)
that minimize or prevent homodimerization, as described, for example, in See, e.g.,
US. Patent Nos. 7,914,796, 8,034,598 and 8,623,618, the disclosures of all ofwhich
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 ofFold are all targets for influencing dimerization of the FOkI cleavage
half-domains where the numbering is with t to the crystal structures lFOKpdb
and 2FOK.pdb (see Wah el al. (1997) Nature 388:97-100) having the sequence shown
below:
Wild type FokI cleavage half domain (SEQ ID NO: 18)
QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVY
GYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRY
VEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITN
CNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF
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 es at positions 490 and 538 ofFold and a second
cleavage half-domain includes mutations at amino acid residues 486 and 499.
Thus, in one embodiment, a on at 490 replaces Glu (E) with Lys
(K), the on at 538 replaces Ile (I) with Lys (K), the mutation at 486 replaced
Gln (Q) with Glu (E), and the on at position 499 replaces Ile (I) with Lys (K).
Specifically, the engineered cleavage half-domains described herein were prepared by
WO 39440
mutating positions 490 (E—>K) and 538 (I—>K) in one cleavage half-domain to
produce an engineered cleavage half-domain designated :IS3 8K” and by
mutating positions 486 (Q—>E) and 499 (I—>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 via the ZFN homodimers is minimized or abolished. See,
e. g., US. Patent Publication No. 2008/0131962, the disclosure of which is
incorporated by reference in its entirety for all purposes. In certain embodiments, the
ered 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) e at position 486 with a Glu (E) residue, the wild type Ile (I)
residue at position 499 with a Leu (L) e 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 ments, the ered cleavage half-
domain comprises mutations at positions 490, 538 and 537 red relative to
wild-type FokI), for ce mutations that replace the wild type Glu (E) residue at
position 490 with a Lys (K) residue, the wild type Ile (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 red 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 ype His (H) residue at position 537 with a Lys (K) residue or a
Arg (R) residue (also referred to as “KIK” and “KIR” domains, tively. See,
e.g., US. Patent Nos. 7,914,796, 8,034,598 and 8,623,618. In other embodiments, the
engineered cleavage half domain comprises the “Sharkey77 (L
7 Sharkey” mutations, and
combinations of the “Sharkey” and “Sharkey” mutations (see Guo el al. (2010) J.
Mol. Biol. :96-107).
Thus, cleavage half domains derived from FokI may comprise a
mutation in one or more of amino acid residues as shown in SEQ ID NO: 18, including
mutations in the dimerization domain as described above, mutations in the catalytic
domain, mutations in other amino acid residues such as phosphate contact residues,
and any ation of mutations selected from mutations in the dimerization
domain, mutations in the catalytic domain, and mutations in other amino acid residues
such as phosphate contact residues. Mutations include substitutions (of a wild-type
amino acid residue with a ent residue), ions (of one or more amino acid
residues), deletions (of one or more amino acid residues), and any combination of
mutations selected from tutions, insertions and deletions. In certain
embodiments, one or more of residues 414-426, 443-450, 467-488, 501-502, 521-531
(numbered relative to SEQ ID NO: 18), and any combination of such residues, are
mutated since these residues are located close to the DNA backbone in a molecular
model of a ZFN bound to its target site described in Miller er a]. ((2007) Nat
Biotechnol 25:778-784). In certain embodiments, one or more residues at positions
416, 422, 447, 448, and 525 are mutated. In certain embodiments, the mutation
comprises a substitution of a wild-type residue with a different residue, for example a
serine (S) residue. In certain embodiments, the FokI cleavage domain of the
nucleases described herein comprise an ELD dimerization domain mutation, a KKR
dimerization domain mutation, a K525S mutation, and any combination selected from
an ELD zation domain on or a KKR dimerization domain mutation and a
K525S mutation.
Engineered cleavage domains described herein can be prepared using
any le method, for example, by site-directed mutagenesis of wild-type cleavage
half-domains (Fok I) as described in US. Patent Nos. 7,888,121, 7,914,796,
8,034,598 and 8,623,618. Furthermore, the cleavage domains described herein may
be fused to a DNA-binding domain (e.g., ZFP) using any le , including,
but not limited to the linkers described in US. Patent Nos. 9,394,531 and 9,567,609.
Alternatively, ses may be assembled in vivo at the nucleic acid
target site using so-called “split-enzyme” technology (see, e.g. US. Patent Publication
No. 2009/0068164). Components of such split enzymes may be expressed either on
separate sion constructs, or can be linked in one open reading frame where the
individual ents are ted, 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.
[0128] Nucleases can be screened for activity prior to use, for example in a
based chromosomal system as described in
20090068164. 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 ce of raffinose, galactose and a ation of
raffinose and galactose and repressed in presence of glucose.
The nuclease(s) as described herein may make one or more double-
ed, one or more single-stranded cuts, and combinations of one or more double-
stranded and one or more single-stranded cuts in the target site. In certain
embodiments, the nuclease comprises a catalytically inactive cleavage domain (e.g.,
FokI, a Cas protein and combinations of FokI and a Cas protein). See, e.g., US.
Patent Nos. 9,200,266, 8,703,489 and Guillinger el al. (2014) Nature h.
32(6):577-5 82. The catalytically inactive cleavage domain may, in combination with
a catalytically active domain act as a nickase to make a single-stranded cut.
Therefore, two nickases can be used in combination to make a double-stranded cut in
a specific region. Additional es are also known in the art, for example,
McCaffery el al. (2016) Nucleic Acids Res. 44(2):e11. doi: 3/nar/gkv878. Epub
2015 Oct 19.
Target Sites
As described in detail above, DNA s 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 BCLl 1A
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 BCLl 1A,
including DNA-binding domains that bind to a sequence within a DNAseI
ensitive site in the BCLl 1A enhancer sequence (e.g., +58) as shown in Table 1.
Engineering methods include, but are not limited to, al design and various types
of selection. Rational design includes, for example, using ses sing
t (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 US. Patent 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., US. Patent Publication No.
2011/0301073.
Exemplary selection methods applicable to DNA-binding domains,
including phage display and two-hybrid systems, are disclosed in US. Patent Nos.
,789,538, 5,925,523, 6,007,988, 453, 6,410,248, 6,140,466, 6,200,759, and
6,242,568, as well as PCT Publication Nos. WO 98/37186, WO 98/53057,
WO 00/27878, WO 01/88197 and UK. Patent No. GB 2,338,237. In addition,
enhancement of g specificity for zinc finger binding s has been
described, for example, in co-owned WO 02/077227.
Selection of target sites, nucleases and methods for design and
construction of fusion proteins (and polynucleotides ng same) are known to
those of skill in the art and described in detail in US. Patent Publication Nos.
2005/0064474 and 2006/0188987, incorporated by reference in their entireties herein.
In addition, as disclosed in these and other references, DNA-binding
s (e.g., fingered zinc finger proteins) and 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. US. Patent Nos.
453, 121 (e.g., “ZC” linker), 6,479,626, 6,903,185, and 7,153,949, US.
Publication No. 2009/0305419) and 2015/0064789. The proteins described herein
may include any combination of suitable linkers between the indiVidual DNA-binding
domains of the protein. See, also, US. Patent No. 8,586,526.
Donors
In certain embodiments, the present disclosure relates to nuclease-
mediated targeted integration of an ous sequence into the genome of a cell
using the BCLl lA 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 , for correction of a
mutant gene, for a combination of deletion of a specified region and 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 mologous ce
flanked by two regions of homology to allow for nt HDR at the location of
interest or can be integrated Via non-homology directed repair mechanisms such as
NHEJ. 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, tinuous regions of homology to cellular
tin, and, for example, lead to a deletion of a BCLl lA enhancer region (or a
fragment therereof) when used as a substrate for repair of a DSB induced by one of
the nucleases described here. Further, for targeted insertion of ces not
normally present in a region of interest, said ces can be present in a donor
nucleic acid molecule and flanked by regions of homology to sequence in the region
of interest.
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 or -stranded, and can be
introduced into a cell in linear or circular form. See, e.g., US. Patent Applicaton
Publication Nos. 047805 and 2011/0207221. 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 ynucleotide residues are added to
the 3’ terminus of a linear molecule and self-complementary oligonucleotides are
optionally ligated to one or both ends. See, for example, Chang er a]. (1987) Proc.
Natl. Acad. Sci. USA84:4959-4963, Nehls el 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, orothioates,
phosphoramidates, and O-methyl ribose or deoxyribose residues. If introduced in
double-stranded form, the donor may e one or more se target sites, for
example, nuclease target sites flanking the transgene to be integrated into the cell’s
genome. See, e.g., US. Patent Publication No. 2013/0326645.
A polynucleotide can be introduced into a cell as part of a vector
molecule having onal 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 xed 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)).
In n embodiments, the double-stranded donor includes sequences
(e.g., coding sequences, also referred to as transgenes) r than 1 kb in length, for
e between 2 and 200 kb, between 2and lOkb (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 ene
sequences, for example, 5’ and/or 3’ to the transgene sequences, for cleavage of the
transgene. The se cleavage ) may be for any nuclease(s). In n
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 s.
The donor is generally ed 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,
AAVSl, etc.). However, it will be apparent that the donor may comprise a promoter,
an enhancer, and combinations of both a promoter and enhancer, for example a
constitutive promoter or an inducible or tissue specific promoter.
The donor molecule may be inserted into an endogenous gene such
that all, some or none of the endogenous gene is expressed. In other ments,
2O 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 Publication
Nos. 2008/02995 80, 2008/0159996 and 2010/00218264.
rmore, 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, polyadenylation signals, and combinations thereof.
The enes carried on the donor sequences described herein may
be isolated from plasmids, cells or other s using standard techniques known in
the art such as PCR. Donors for use can include varying types of gy, 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).
The double-stranded donor polynucleotides described herein may
include one or more non-natural bases, one or more backbones, and ations of
one or more non-natural bases and one or more nes. 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.
The exogenous (donor) polynucleotide may comprise any sequence of
interest (exogenous sequence). Exemplary exogenous sequences include, but are not
limited to any polypeptide coding ce (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 d
to, sequences encoding proteins that mediate antibiotic resistance (e.g., ampicillin
resistance, kanamycin resistance, neomycin resistance, G418 resistance, puromycin
resistance, ycin resistance, blasticidin resistance), sequences encoding colored
or fluorescent or scent proteins (e.g., green fluorescent protein, enhanced green
fluorescent protein, red fluorescent protein, luciferase), and proteins which mediate
enhanced cell growth, gene amplification (e.g., dihydrofolate reductase) and
ations of enhanced cell growth and gene amplification. Epitope tags include,
for example, one or more copies of FLAG, His, Myc, tandem affinity purification
(TAP), HA, biotinylatable peptide, or any able amino acid sequence.
[0144] In a preferred embodiment, the ous ce (transgene)
comprises a polynucleotide encoding any polypeptide of which expression in the cell
is desired, including, but not limited to antibodies, antigens, enzymes, ors (cell
surface or nuclear), hormones, lymphokines, cytokines, er polypeptides, growth
factors, and functional fragments of any of the above. The coding sequences may be,
for example, cDNAs.
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, ing but not limited to any of the following genetic diseases:
achondroplasia, achromatopsia, acid maltase deficiency, adenosine ase
deficiency (OMIM No.102700), adrenoleukodystrophy, aicardi syndrome, alpha-l
antitrypsin deficiency, alpha-thalassemia, androgen itivity 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., GMl), hemochromatosis, the hemoglobin C mutation in the 6th
codon of lobin (HbC), hemophilia, Huntington’s disease, Hurler Syndrome,
hypophosphatasia, Klinefleter syndrome, Krabbes Disease, Langer-Giedion
Syndrome, leukocyte adhesion deficiency (LAD, OMHVI No. 116920),
leukodystrophy, long QT syndrome, Marfan syndrome, Moebius syndrome,
mucopolysaccharidosis (MPS), nail patella syndrome, nephrogenic diabetes insipdius,
neurofibromatosis, Neimann-Pick e, enesis ecta, 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 e,
Thrombocytopenia Absent Radius (TAR) syndrome, Treacher Collins syndrome,
trisomy, tuberous sclerosis, Turner’s me, urea cycle disorder, von Hippel-
Landau disease, Waardenburg syndrome, Williams syndrome, Wilson’s disease,
Wiskott-Aldrich syndrome, X-linked proliferative syndrome (XLP, OMIM
No. 308240).
Additional ary diseases that can be treated by targeted
integration include acquired immunodeficiencies, lysosomal storage diseases (e.g.,
Gaucher’s e, GMl, Fabry disease and Tay-Sachs disease),
mucopolysaccahidosis (e.g. ’s disease, Hurler’s disease), hemoglobinopathies
(e.g., sickle cell disease, HbC, d-thalassemia, B-thalassemia) and hemophilias.
In certain embodiments, the exogenous sequences can comprise a
marker gene (described , allowing selection of cells that have undergone
ed integration, and a linked ce encoding an additional functionality.
Non-limiting examples of marker genes include GFP, drug selection marker(s) and
the like.
Additional gene sequences that can be inserted may include, for
example, wild-type genes to replace d 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 d. The ype copy may be inserted at the
endogenous locus, or may alternatively be targeted to a safe harbor locus.
Construction of such expression cassettes, following the teachings of
the present specification, utilizes methodologies well known in the art of lar
biology (see, for e, 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).
Furthermore, although not required for expression, exogenous
ces may also transcriptional or translational regulatory sequences, for example,
promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding
2A peptides, polyadenylation signals, and combinations of 2A polypeptides and
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
tes).
[0151] Targeted insertion of non-coding nucleic acid sequence may also be
achieved. ces encoding antisense RNAs, RNAi, shRNAs and micro RNAs
s) may also be used for targeted insertions.
In additional embodiments, the donor nucleic acid may comprise non-
coding sequences that are specific target sites for additional nuclease s.
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
st. In this way, reiterative integrations of donor les may be generated
allowing for trait stacking at a particular locus of interest or at a safe harbor locus.
Delivery
The nucleases as described herein (Table l), polynucleotides ng
these nucleases, donor cleotides and compositions comprising the proteins,
polynucleotides, and combinations of proteins and polynucleotides described herein
may be delivered in vivo or ex vivo by any suitable means into any cell type.
[0154] Suitable cells include eukaryotic (e.g., animal) and prokaryotic cells
and eukaryotic and prokaryotic cell lines. Non-limiting examples of such cells or cell
lines generated from such cells include COS, CHO (e.g., CHO-S, CHO-Kl, CHO-
DG44, XBll, KX, CHOKISV), VERO, MDCK, w13 8, v79,
B14AF28-G3, BHK, HaK, NSO, SP2/O-Agl4, HeLa, HEK293 (e. g., HEK293-F,
HEK293-H, HEK293-T), and perC6 cells as well as insect cells such as
Spodopterafuglperda (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,
nic stem cells, induced pluripotent stem cells, poietic stem cells,
neuronal stem cells and mesenchymal stem cells.
Methods of delivering nucleases as described herein are described, for
example, in Us. Patent 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, 219, and 7,163,824, the
disclosures of all of which are incorporated by reference herein in their entireties.
Nucleases, donor constructs, and combinations of nucleases and donor
constructs as bed herein may also be delivered using vectors containing
sequences encoding one or more of the ZFN(s), described . Any vector systems
may be used including, but not limited to, plasmid vectors, retroviral vectors,
lentiviral s, adenovirus vectors, poxvirus vectors, virus vectors and
associated virus vectors, etc. See, also, US. Patent Nos. 6,534,261, 6,607,882,
978, 6,933,113, 6,979,539, 219, and 7,163,824, incorporated by nce
herein in their ties. 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 ses and a donor construct are introduced into the cell, the nucleases,
donor polynucleotide, and combinations of nucleases and 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, one or more donor constructs, and combinations of one or more nucleases
and one or more donor constructions. Conventional viral and non-viral based gene
transfer methods can be used to introduce nucleic acids encoding nucleases, donor
constructs, and combinations of nucleases and donor constructs in cells (e.g.,
mammalian cells) and target tissues. Non-viral vector delivery s 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 s 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):829-83 9,
Rossi et al (2007) Nature Biotech. :1444-1454 as well as general gene delivery
nces such as on, 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 , 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).
Methods of non-viral delivery of nucleic acids include electroporation,
lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes,
polycation or lipid:nucleic acid conjugates, naked DNA, ial virions, membrane
ation, and agent-enhanced uptake of DNA. Sonoporation using, e.g., the
Sonitron 2000 system Mar) can also be used for delivery of nucleic acids.
Additional exemplary nucleic acid delivery systems include those
provided by Amaxa Biosystems ne, y), Maxcyte, Inc. (Rockville,
Maryland), BTX Molecular ry Systems (Holliston, MA) and Copernicus
Therapeutics Inc., (see for example 336). Lipofection is described in e.g,
US. Patent Nos. 5,049,386, 4,946,787, and 4,897,355) and lipofection reagents are
sold commercially (e.g, TransfectamTM and LipofectinTM). Cationic and neutral
lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides
include those of Felgner, W0 91/17424, W0 91/16024.
[0159] 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. 5382-3 89 (1994), Remy et al.,
Bioconjugate Chem. 5:647-654 , Gao et al., Gene Therapy 2:710-722 (1995),
Ahmad et al., Cancer Res. 52:4817-4820 (1992), US. Pat. Nos. 4,186,183, 4,217,344,
4,235,871, 4,261,975, 054, 4,501,728, 4,774,085, 028, and 4,946,787).
Other lipid:nucleic acid complexes e those sing novel cationic lipids,
novel pegylated lipids, and combinations of novel cationic lipids and novel pegylated
lipids (see e.g. US. ional Patent Application Nos. 62/432,042 and 62/458,373).
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 el al. (2009) Nature Biotechnology 27(7):643).
The use of RNA or DNA viral based systems for the delivery of
c acids encoding engineered ZFPs, TALEs and CRISPR/Cas systems take
advantage of highly d 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, lentiviral,
adenoviral, adeno-associated, vaccinia and herpes simpleX virus vectors for gene
transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and
associated virus gene transfer methods, often resulting in long term expression
of the inserted transgene. Additionally, high uction efficiencies have been
observed in many different cell types and target tissues.
2O [0162] The tropism of a irus can be altered by incorporating foreign
envelope proteins, expanding the potential target population of target cells. iral
vectors are iral s that are able to transduce or infect non-dividing cells and
typically produce high viral titers. Selection of a retroviral gene er system
depends on the target tissue. iral vectors are comprised of ting long
terminal s with packaging capacity for up to 6-10 kb of n 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 (SIV), human immunodeficiency virus (HIV), and
ations thereof (see, e.g., Buchscher er al., J. Virol. 66:2731-2739 (1992),
Johann el al., J. Virol. 66:1635-1640 (1992), Sommerfelt el al., Virol. 176:58-59
(1990), Wilson el al., J. Virol.63:2374-2378 (1989), Miller el al., J. Virol. 65:2220-
2224 (1991), PCT/US94/05700).
In applications in which transient expression is preferred, adenoviral
based systems can be used. Adenoviral based s are capable of very high
transduction efficiency in many cell types and do not e 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 c acids and peptides, and for in vivo and ex vivo gene
therapy procedures (see, e.g., West el al., Virology 160:38-47 (1987), US. Patent 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
s are described in a number of publications, including US. Pat. No. 5,173,414,
Tratschin el al., Mol. Cell. Biol. 53251-3260 (1985), Tratschin, el al., Mol. Cell. Biol.
4:2072-2081 , at & Muzyczka, PNAS 81 :6466-6470 , and
Samulski el al., J. Virol. 63:03822-3828 (1989).
[0164] 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.
pLASN and MFG-S are examples of retroviral vectors that have been
used in clinical trials (Dunbar el al., Blood 85:3048-305 (1995), Kohn el al., Nat.
Med. 1:1017—102(1995), Malech eial, PNAS 94:22 12138 (1997)).
PA3 l7/pLASN was the first therapeutic vector used in a gene therapy trial. e el
al., Science 270:475-480 (1995)). Transduction efficiencies of 50% or greater have
been observed for MFG-S packaged vectors. (Ellem el al., Immunol Immunolher.
44(1): 10-20 (1997), f el al., Hum. Gene Ther. 1:111-2 (1997).
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 ene
sion 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 el al., Lancet 351 :91 17 1702-3 (1998), Keams el al., Gene Ther.
9748-55 (1996)). Other AAV serotypes, including AAVl, AAV2, AAV3, AAV4,
AAVS; AAV6; AAV7; AAV8; AAV9 and AAVrh. 10 and any novel AAV serotype
can also be used in accordance with the present invention.
Replication-deficient inant 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 Ela; Elb; or
E3 genes; subsequently the replication defective vector is ated in human 293
cells that supply deleted gene function in trans. Ad vectors can transduce multiple
types of tissues in viva; 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 cleotide
therapy for mor immunization with intramuscular injection (Sterman el al.,
Hum. Gene Ther. 7: 1083-9 (1998)). Additional examples of the use of adenovirus
vectors for gene transfer in clinical trials include cker el al., Infection 24:1 5-
; n el al., Hum. Gene Ther. 9:7 1083-1089 (1998); Welsh el al., Hum.
Gene Ther. 2:205-18 (1995); Alvarez el al., Hum. Gene Ther. 5:597-613 (1997); Topf
el 61]., Gene Ther. 513 (1998); Sterman el al., Hum. Gene Ther. -1089
(1998).
Packaging cells are used to form virus particles that are capable of
infecting a host cell. Such cells include HEK293 and Sf9 cells; which can be used to
package AAV and adenovirus; and \VZ cells or PA3 17 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 ing and subsequent integration into a host
(if applicable); other viral sequences being replaced by an expression cassette
encoding the protein to be expressed. The g viral functions are supplied in
trans by the packaging cell line. For example; AAV vectors used in gene therapy
lly only s 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 ng 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. In some embodiments, AAV is produced
using a baculovirus expression system (see e.g. US. Patent Nos. 6,723,551 and
7,271,002).
Purification of AAV particles from a 293 or baculovirus system
typically involves growth of the cells which produce the virus, ed by collection
of the viral les from the cell supernatant or lysing the cells and collecting the
virus from the crude lysate. AAV is then purified by methods known in the art
including ion exchange chromatography (e.g. see US. Patent Nos. 7,419,817 and
6,989,264), ion exchange chromatography and CsCl density centrifugation (e.g. PCT
publication 094198A10), immunoaffinity chromatography (e.g.
W02016128408) or purification using AVB Sepharose (e.g. GE Healthcare Life
Sciences).
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
sing 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 el al., Proc. Natl. Acad. Sci. USA -
9751 , reported that Moloney murine leukemia virus can be modified to express
2O 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 n sing a ligand for the cell-
surface receptor. For example, filamentous phage can be ered to display
antibody nts (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
ered to contain specific uptake sequences which favor uptake by specific target
cells.
[0171] 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 dual patient (e.g., lymphocytes, bone
marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells,
followed by antation of the cells into a patient, usually after selection for cells
which have incorporated the vector.
Vectors (e.g., retroviruses, adenoviruses, mes, etc.) containing
nucleases, donor constructs, and combinations of ses and donor ucts 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 d to, injection, infusion, topical application and
electroporation. Suitable methods of administering such c 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 ition, a particular route can often provide a
more immediate and more effective reaction than another route.
Vectors le for introduction of cleotides (e.g. nuclease-
encoding, double-stranded donors, and combinations of nuclease-encoding and
double-stranded donors) described herein include tegrating lentivirus vectors
. See, for example, Ory et al. (1996) Proc. Natl. Acaa’. Sci. USA 93:11382-
11388, Dull et al. (1998) J. Virol.72:8463-8471, Zuffery et al. (1998) J. Viral.
72:9873-9880, Follenzi et al. (2000) Nature Genetics 25:217-222, US. Patent
Publication No. 2009/0117617.
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 ceutical compositions available, as described below (see, e.g.,
Remington’s Pharmaceutical Sciences, 17th ed., 1989).
It will be apparent that the nuclease-encoding sequences and donor
constructs can be red using the same or different systems. For example, the
nucleases and donors can be d 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 or intramuscular
injection). The vectors can be delivered simultaneously or in any sequential order.
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 ion 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 retrograde injection h
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 el al., (1994) Nature Genetics, 6335-341.
The effective amount of se(s) and donor to be administered will
vary from patient to t and according to the therapeutic polypeptide of interest.
ingly, effective s are best determined by the physician administering
the compositions and appropriate dosages can be determined readily by one of
ry skill in the art. After allowing sufficient time for ation 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 uent administrations if
necessary. Subsequent administrations may be administered at le intervals,
ranging from daily to ly 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 el al., (1995) Human Gene Ther, 6: 1391-1401.
Formulations for both ex vivo and in vivo administrations include
suspensions in liquid or emulsif1ed liquids. The active ingredients often are mixed
with excipients which are pharmaceutically acceptable and compatible with the active
ient. Suitable ents 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
Also described herein are cells and cell lines in which an endogenous
BCLl lA enhancer sequence is modified by the ses described herein (Table l).
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 BCLl lA ce may comprise indels.
The modification is preferably at or near the se(s) binding
site(s), cleavage ) and combinations of binding site(s) and cleavage site(s), for
example, within 1-300 (or any value therebetween) base pairs upstream or
ream of the site(s) of cleavage, more ably within 1-100 base pairs (or any
value therebetween) of either side of the binding site(s), cleavage site(s), or binding
site(s) and ge site(s), even more preferably within 1 to 50 base pairs (or any
value etween) on either side of the binding site(s), cleavage site(s), or binding
site(s) and cleavage site(s). In certain embodiments, the modification is at or near the
“+58” region of the BCLl lA enhancer, for example, at or near a nuclease g site
shown in any of the first column of Table l.
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),
tic 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 sor cell). Non-limiting
examples other cell lines including a modified BCLl lA sequence include COS, CHO
(e.g., CHO-S, CHO-Kl, CHO-DG44, CHO-DUXBI 1, CHO-DUKX, CHOKISV),
VERO, MDCK, w13 8, v79, B14AF28-G3, BHK, HaK, NSO, SP2/O-Agl4, HeLa,
HEK293 (e.g., HEK293-F, HEK293-H, -T), and perC6 cells as well as insect
cells such as Spodopterafugiperda (Sf), or fungal cells such as Saccharomyces, Pichia
and Schizosaccharomyces.
The cells as bed herein are useful in treating or preventing a
er, for example, by ex vivo therapies. The nuclease-modif1ed cells can be
expanded and then reintroduced into the patient using standard techniques. See, e.g.,
Tebas er a]. (2014) New Eng JMed 370(10):90l. In the case of stem cells, after
infusion into the subject, in vivo differentiation of these precursors into cells
expressing the onal 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.
Any of the modified cells or cell lines disclosed herein may show
increased expression of gamma globin. Compositions such as pharmaceutical
compositions sing the genetically modified cells as described herein are also
provided
Applications
[0184] 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 e or a thalassemia. Thus,
the methods and compositions provide for the treatment or prevention of such genetic
es. 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. ng at least one
globin (e. g., 0t globin, y globin, B globin and combinations thereof), may be inserted
into a cell (e.g., into an endogenous BCLl lA enhancer sequence using one or more
nucleases as described herein) to provide the globin proteins def1cient or lacking in
the cell and thereby treat a c disease, e.g., a hemoglobinopathy, caused by faulty
globin expression. Alternatively or in addition, genomic editing with or t
administration of the appropriate donor, can correct the faulty endogenous gene, e.g.,
correcting the point mutation in 0(- or B-hemoglobin, to restore expression of the gene
or treat a genetic disease, e.g. sickle cell disease, knock out or tion
(overexpression or repression) of any direct or indirect globin regulatory gene (e.g.
inactivation of the y globin-regulating gene BCLl ler the BCLl lA-regulator KLFl).
Specifically, the methods and compositions of the invention have use in the treatment
or tion of hemoglobinopathies.
WO 39440
The nucleases of the invention are targeted to the BCLl lA enhancer
region, known to be required for the expression of BCLl lA during opoiesis,
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.
The following Examples relate to exemplary embodiments of the
present disclosure in which the nuclease comprises a zinc finger nuclease (ZFN). It
will be appreciated that this is for purposes of if1cation only and that other
nucleases can be used, for example TtAgo and CRISPR/Cas s, homing
endonucleases ucleases) with engineered DNA-binding domains, fusions of
naturally ing of engineered homing cleases (meganucleases)
DNA-binding domains, including combinations of homing endonucleases
(meganucleases) with engineered DNA-binding domains and fusions of naturally
occurring of ered homing endonucleases (meganucleases) DNA-binding
domains, and heterologous cleavage domains, fusions of meganucleases and TALE
proteins, including combinations of heterologous cleavage domains and fusions of
meganucleases and TALE proteins.
EXAMPLES
Example 1: Assembly of Zinc Finger Nucleases
ZFNs were assembled against the human BCLl lA gene and activity
was tested by deep cing analysis ofDNA isolated from transfected cells as
described below. ZFNs specific for the +58 region of the enhancer region were made
as described. ZFN pair 51857/51949 has been described previously (see WO
2016/183298).
Example 2: Off target is
To analyze off target cleavage by the ZFN pairs, a two stage unbiased
specif1city is was performed. In the first stage, (Figure 1), candidate off-target
sites for each ZFN were identified via an oligonucleotide duplex integration site assay
using a ure similar to that described by Tsai er a]. ((2015), Nat Biotechnol
33(2): 187-197. doi: lO. lO38/nbt.3 1 17).
The oligonucleotide duplex integration site assay is based on the
observation that co-introduction of a nuclease and a short t of duplex DNA
into a target cell results in dplex integration during repair of a fraction of genome
cleavage events via the NHEJ DNA repair pathway (Orlando er al., (2010), Nucleic
Acids Res, 38(15)e152. doi: 10.1093/nar/gkq512, Gabriel el al., (2011), Nat
Biotechnol. 2011 Aug 7,29(9):816-23. doi: 10.1038/nbt.l948, Tsai el al., ibid). Upon
integration the duplex es a permanent tag of the cleavage event. Sites of
integration are then identified via ligation of an oligonucleotide adaptor to sheared
genomic DNA, followed by 2 rounds of 25 cycles of nested PCR, and deep
sequencing of the resulting donor-genome junctions. This assay allows for evaluation
of all potential integration sites within the genome.
The integration site assay was med in K562 cells to maximize
donor delivery, ZFN expression, and donor integration. Moreover as K562 cells
divide quickly (doubling time approximately 24 hours) they are expected to impose
minimal epigenetic restrictions on the ability ofZFNs to cleave cellular targets. Cells
(2 x 105) were electroporated with 0.47 ug of oligonucleotide duplex donor and 400
ng of each ZFN-encoding mRNA using an Amaxa e and settings optimized for
maximal on-target activity of the ZFNs. Four replicate samples were prepared for
each ation of oligo and mRNA. On day 7 post-transfection, genomic DNA
was isolated for each sample (Qiagen DNeasy Blood and Tissue Kit) and 400 ng
(133000 d genomes) was used as input for the amplification protocol outlined in
Figure 1. Samples were then sed essentially as described (Tsai el al. ibid).
Final products were pooled, quantified, and sequenced on a MiSeq Instrument
(Illumina) using a v2 300 cycle cing kit with paired-end 150 bp reads and 8
bp/l6 bp dual index reads to detect the sample barcodes on each end of the amplicon.
To generate a list of candidate off target sites, sequencing data were
filtered for correct priming sequence, followed by trimming of r sequences and
mapping to the genome. Next, junction coordinates were mapped, and the duplex-
genome on, as well as the position of the break caused by DNA shearing, were
used to identify ct integration events. Integration events were then processed to
identify clusters of integrations in close proximity within the genome (minimum of 4
ct integration events within 100bp of each other, summed across all replicates).
Clusters residing on contigs that were unmappable in the hg38 ly (zle. chrUn in
hg3 8) were removed from further analysis. rs mapping to repetitive loci
(median of three or more hits to the genome across all sequences in a cluster) were
also removed as prior experience has shown these to be amplification artifacts.
Remaining clusters were scored as ate ZFN cleavage sites if they were derived
from at least 2 ate ZFN treated samples (of 4 total) and exhibited E 5 fold
excess of integration events in ZFN treated samples versus controls. Candidate
cleavage sites were ranked by the total number of unique integrations in the ZFN
treated samples.Candidate loci identified via this analysis are provided in Figure 2 for
ZFN pair 51857/51949, ranked by integrant count.
Example 3: Optimization of ZFNs
To decrease off target cleavage, a strategy for nuclease optimization in
which nonspecific phosphate contacts are selectively d to bring about global
suppression off-target cleavage (Guilinger et a]. (2014) Nut Methods. 11(4):429-35.
doi: 10.1038/nmeth.2845, Kleinstiver et a]. (2016) Nature 529(7587):490-5. doi:
10.1038/nature16526, Slaymaker et a]. (2016) Science) 351(6268):84-8. doi:
.1126/science.aad5227) was d (see US. ional Application Nos.
62/443,981 and 62/378,978). Amino acid substitutions were made at a key position
within the zinc finger ork that interacts with the phosphate backbone of the
DNA (Pavletich and Pabo, (1991) Science 252(5007):809-17, Elrod-Erickson et a].
2O (1996) Structure 4(10):1171-80) (Figure 3A-3B) as well as at a single on in the
right ZFN FokI domain also predicted to make a phosphate contact e 3C).
Specificity was further improved by allowing independent expression
of each ZFN from two separated mRNAs, which enables optimization of delivery
ratios. These efforts yielded optimized ZFN pairs that are highly related to the
original one, ing by substitutions that decrease the energetics of interaction with
the DNA phosphate backbone but that lly or do not impact sequence specific
base recognition. Consistent with this, the integration site assay d 455 loci for
potential targets of ZFN cleavage for the original 51857/51949 pair. For the
optimized pair, a much smaller number of loci were identified for r examination
as potential targets of ZFN cleavage (72 total) by this analysis. For both pairs, the
intended target within the BCL11A enhancer was the top ranked locus. Moreover a
much higher fraction of integration events was noted at the BCL11A enhancer for the
optimized pair, consistent with its greater specificity.
It is important to note that in defining the sequence data processing
pipeline, key parameters were chosen conservatively, to err on the side of including as
many ate off target loci as feasible instead of filtering them out. This was done
to ensure that every locus that might represent a bona fide cleavage site for the
zed ZFNs would be identified and tested in follow-up indel studies, even at the
cost of accepting a much greater number that would become false positives. It was
expected that the first stage of is would yield a large set of candidate loci for
each ZFN pair, of which the large majority (particularly for the optimized ZFNs)
would not represent true off-target cleavage sites but rather background events that
would prove negative for cleavage in follow-up indel studies.
In the second stage of analysis, candidate off-target loci identified via
the integration site assay were screened for evidence of modification (e.g., the
presence of indels) in ZFN-treated CD34+ HSPC.
In particular, human CD34+ HSPC derived from mobilized peripheral
blood were treated with the original and optimized ZFN pairs using clinical scale and
clinical conditions for RNA transfection (l20 ug/mL of mRNA for the original ZFN
pair and 100 ug/mL of mRNA for the optimized pair). Genomic DNA was isolated 2
days post-transfection, followed by PCR amplification of candidate off-target loci and
deep cing to quantify indel levels. For both the original and optimized ZFN
2O pairs, the same set of 137 candidate off-target loci were screened at this step, along
with a r number of candidate off-target sites that had been identified via other
s in earlier studies with the al ZFNs.
The s showed that the optimized ZFNs are markedly more
specific than the original pair. This is apparent not only from the number of loci that
were scored ve for evidence of ZFN ge (52 for the original pair vs 3 for
the optimized pair), but also from the ed indel levels, which for the optimized
pair were much lower. Figure 4 shows plots of indel values at every locus exhibiting
evidence of ZFN cleavage in this study (note log scale of y aXis). Aggregating off-
target indels across all such loci indicates a reduction in off-target activity of 300 fold
(46.5% aggregate off-target indels for the original pair, vs 0.15% off-target indels for
the optimized pair). This reduction in off-target activity was achieved without any
loss in activity at the intended target site (72.5% indels for the original pair vs 81.9%
for the optimized ZFNs). In these studies, the original pair (or parental pair) was
51857/51949, while the optimized ZFN pair was 63014/65722 (see below).
The nuclease s are shown below in Table 1:
Table 1: ZFN pairs specific for +58 BCL11A enhancer region
Design
[Helix Sequence, ST
—-[Mutationsto finger backbone] Fok
————————
Left artner
51857 DQSNLRA RNFSLTM STGNLTN TSGSLTR DQSNLRA AQCCLFH
aaAGCAACtG T T (SEQ ID (SEQ ID T T
TTAGCTTGCA NO: 21) NO: 22)
Ctagacta
(SEQ ID L“ U
NO:1)
63014 DQSNLRA RNFSLTM STGNLTN TSGSLTR A AQCCLFH
ACtG T T (SEQ ID (SEQ ID T T
TTAGCTTGCA NO:2l) NO:22)
(SEQ ID Li] L“ U
NO:1)
65459 DQSNLRA RNFSLTM STGNLTN TSGSLTR DQSNLRA H
aaAGCAACtG T T (SEQ ID (SEQ ID T T
TTAGCTTGCA NO: 21) NO: 22)
Ctagacta
(SEQ ID le4Qm5 none none none L“ U
NO:1)
Rio_ht Partner
51949 QKAHLIR QKGTLG O
caCAGGCTCC T (SEQ ID (SEQ I
AGGAAGGg:t : NO:25) NO:26) IE]
tggcctc:
(SEQ ID
NO:2)
65722 QKAHLIR QKGTLG
caCAGGCTCC T (SEQ ID (SEQ I
AGGAAGGg:t : NO:25) NO:26)
tggcctc:
(SEQ ID
NO:2)
65526
QKAHLIR QKGTLGI
caCAGGCTCC ;: (SEQ ID (SEQ I
AGGAAGtht : NO:25) NO:26)
tggcctc:
(SEQ ID
NO:2)
65549 QKAHLIR QKGTLG
caCAGGCTCC T (SEQ ID (SEQ I
AGGAAGGg:t : NO:25) NO:26)
tggcctc:
(SEQ ID
NO:2)
WO 39440
65550 RNDHRTT QKAHLIR QKGTLGH QDLSR RRDNLHS
caCAGGCTCC SEQ ID SLQ ID (SEQ I SLQ ID SLQ ID
AGGAAGGg:t NO: 24) NO: 25) NO:26) NO: 26) NO: 27)
tggcctc' KKR
(SLQ ID K5258
NO: 2)
Table 1 shows characterizing information pertaining to each ZFN.
Starting from the left, the SBS number (e.g. 51857) is yed with the DNA target
that the ZFN binds to displayed below the SBS number. Next are shown the amino
acid recognition helix designs for fingers 1-6 or 1-5 (subdivided column 2 of Table 1).
Also shown in Table 1 under the appropriate helix designs are mutations made to the
ZFP backbone sequences of the indicated finger, as described in US. Provisional
Patent Application Nos. 62/378,978 and 62/443,981. In the notation used in Table 1,
“Qm5” means that at position minus 5 ive to the helix which is numbered -1 to
+6) of the indicated finger, the arginine at this on has been replaced with a
glutamine (Q), while “le4” means that the arginine (R) normally present in
position minus 14 has been replaced with a glutamine (Q). “None” indicates no
changes outside the recognition helix region. Thus, for example, SB S# 63014
includes the Qm5 mutation in fingers 1, 3 and 5 while fingers 2, 4 and 6 do not have
mutations to the zinc finger backbone (e.g., the zinc finger ce outside the
recognition helix ).
Finally, the right-most column of Table 1 shows the linker used to link
the DNA binding domain to the FokI ge domain (e.g., “L7c5”
(LRGSISRARPLNPHP (SEQ ID NO:5), as described for example in US. Patent No.
9,567,609) is displayed on top line of the column, with the sites of the FokI phosphate
contact mutations and dimerization mutations shown in the box below the linker
designation. In specifics, indicated on top line of the Fok mutants box is the type of
mutation found in the dimerizing domain (e.g., ELD or KKR as described for example
in US. Patent No. 8,962,281). Below the dimerization mutant designations is shown
any mutations t in the FokI domain made to remove a non-specific ate
contact shown on the bottom (e.g. K525S or R416S where serine residues at amino
acid ons 525 or 416 have been substituted for either a lysine or arginine,
respectively as described in US. Provisional Patent Application Nos. 62/378,978 and
62/443,981). Thus, for example, in SBS# 63014, the linker is an L7c5 linker and the
FokI cleavage domain includes the ELD dimerization mutants and no phosphate
contact mutations. Further, for SB S# 65722, the linker is an L0 linker (LRGSQLVKS
(SEQ ID NO:6), also referred to as the ‘standard’ linker, see US. Patent No.
9,567,609) and the FokI cleavage domain includes the KKR dimerization mutations
and the K525S FokI phosphate contact mutation.
All ZFNs were tested for functionality (cleavage activity as determined
by assaying for indels as described in Example 4 below) and found to be active.
Furthermore, in order to determine which ZFN designs were the most
specific, indel analyses of known sites of off-target ge by the original ZFN pair
were performed in ZFN-treated CD34+ HSPC. To accomplish this, human CD34+
HSPC derived from mobilized peripheral blood were treated with the original and
optimized ZFN pairs using clinical conditions and mRNA concentrations (120 ug/mL
for the original ZFN pair and 100 ug/mL for the optimized pair). Genomic DNA was
isolated 2 days post-transfection from these cells and untreated controls, followed by
PCR amplification of each candidate locus and deep sequencing to quantify indel
levels.
Modification levels at each locus were determined by paired-end deep
sequencing on an Illumina MiSeq using a 300 cycle cartridge. Paired sequences were
merged, adaptor d via SeqPrep filtered for a quality score of 215 across all
bases, and then mapped to the human genome (hg38 ly). ces that
mapped to an incorrect locus were discarded. Sequences shorter than the wild-type
on by >70 bp or >70% were removed in order to ze primer-dimer
products. A Needleman-Wunsch alignment (Needleman and Wunsch, (1970), JMol
Biol 48(3):443-53)) was med n the target on and each MiSeq read
to map . Indels in aligned sequences were defined as described in Gabriel el al.
2011 (ibid) except that indels lbp in length were also accepted to avoid undercounting
real events. Note that a fraction of loci either did not amplify or did not sequence, or
were rejected from analysis due to high background (>l% ation in control
samples) or insufficient sequencing depth (<10000 reads). The results of this analysis
and comparison to the ‘parent’ 51857/51949 ZFN pair are provided below in Table 2.
WO 39440
Table 2: Off target cleavage analysis
ZFN dimer -- Off-target: # of loci # indel-positive OT loci
Left Right pg BCLl 1A target PCR’d Analyz- P<0.05 manual In parent?
RNA % able Capture/confirmed
L:R '
63014 65526 60: 15 81.2
63014 65549 60: 60 80.0
63014 65550 60: 60 79.8 1/1
65459 65526 60: 15 76.9 2/2
Example 4: Activity of ZFNs in human CD34+ cells
For in vitro testing, the nucleases were tested in CD34+ cells. ZFNs
were supplied as mRNAs, where the mRNAs were made in vitro as follows: plasmids
comprising the genes ng the ZFN are ized and used for in vitro mRNA
transcription using the mMessage mMachine® T7 Ultra Kit (Ambion/Applied
Biosystems). The mRNA was then purified using an RNeasy® mini kit n).
[0205] CD34+ cells were isolated from mobilized peripheral blood and
maintained in X-VIVO 10 medium supplemented with penicillin, streptomycin and
glutamine as well as StemSpan CCl 10 and incubated at 37°C and 5% C02. Cells
were transfected 48 hours post-isolation or post-thaw. A small aliquot was mixed 1:1
with trypan blue solution 0.4% (w/V) in PBS (Corning) and the cell numbers were
determined on a TC20 Automated Cell Counter (Bio-Rad).
For large scale transfections, cells were washed with MaXCyte
Electroporation Buffer te) and re-suspended at 3 to 567 cells per mL in
Electroporation buffer in 100 pL. Typically, mRNA concentrations between 60
pg/mL and 120 pg/mL were used to screen candidate ZFN sets. Cells were then
grown in growth media at 3e6 cells per mL for 18 hours at 300C and then diluted to
1e6 cells per mL for an additional 24 hours at 37°C. For ination of cleavage
activity, genomic DNA was isolated 2-3 days post-transfection, and the level of gene
modification at the BCLl 1A er locus was measured Via deep sequencing on a
MiSeq sequencer (Illumina).
The ZFN pairs from Table 1 were tested in CD34+ cells and the
activity results are shown below in Table 3.
Table 3: Activity of ZFN pairs against BCL11A target
GFP control 0.07
In addition to analyzing the nuclease actiVity in CD34+ cells prior to
erythroid differentiation, edited cells were also differentiated in vitro into erythroid
cells. The protocol followed was based on tana el al., ((2011) Blood 120
(15):2945-53). In brief, the ol below was followed:
Day 0 to Day 7: 4x104 CD34+ cells were cultured at a y of
2X104 /mL in differentiation medium (EDM) (Iscove’s Modified Dulbecco’s Medium
[HVIDM], 330 ug/mL Transferrin ,10 ug/mL Human Insulin, 2 U/mL Heparin sodium,
5% Human AB+ plasma) in the presence of 10‘6 M hydrocortisone, 100 ng/mL stem
cell factor (SCF), 5 ng/mL IL 3, and 3 IU/mL erythropoietin (EPO).
Day 4: Cells resuspended in fresh EDM containing
SCF, IL-3, EPO, and hydrocortisone.
Day 7 to Day 11: Cells were resuspended at a density of 1.5 X 105
cells per fresh mL ofEDM supplemented with SCF and EPO.
Day 11 to Day 21: On day 11, cells were replated at 1 X 106 /mL in
fresh EDM supplemented with EPO. Cells were subsequently replated in this same
media at 5 X 106 /mL on day 14. Growth plateaus during this period of time between
day 14 to 18, when cell Viabilities began to drop until termination of the cultures at
day 21.
Cell counts were taken at the time of seeding and throughout the
differentiation by measuring Acridine Orange positivity and Propidium Iodide
exclusion (AOPI) using a Nexcelom Bioscience Cellometer K2 with the AOPI
Erythroid Assay mode with fluorescence channel 1 (A0) set to 700 milliseconds and
fluorescence channel 2 (PI) set to 5000 milliseconds.
The tage of enucleated cells was determined at day 21 of the
entiation using the ing protocol. The enucleation rate was comparable
among untransfected controls and ZFN-transfected samples with tages 59-63%
from these two groups:
.°°.\‘.O‘.U‘.-'>P°.N.H Cell count
100,000 cells, spin down at 450 X g, 5 min, RT.
Resuspend in 50 uL PB S-B SA + l uL ITC (DAKO).
Stain for 15 min in fridge.
Add 1 mL PBS-B SA, vortex, spin down.
Resuspend in 250 uL of PB S-BSA-NucRed (2 drops NucRed per mL).
Acquire on FACS Canto using the APC channel for NucRed.
Nucleated erythroid cells will be in the GlyA positive NucRed negative/low
fraction and erythroblasts will be in the double GlyA-NucRed positive
fraction.
[0215] BCLl lA gene modification was measured by MiSeq deep sequencing
in DNA s harvested a) 48 hours after electroporation b) on the day of thawing
the cells, at the time when the in vitro differentiation was started and c) at day 14 of
the in vitro erythroid differentiation. While the entiation was performed for
21 days, the day 14 timepoint for DNA analysis was chosen since it is prior to
enucleation of a large on of the erythroid cells which results in a loss ofDNA
recovery. The observed modif1cation percentages at the BCLl lA enhancer are listed
in Table 4 together with details of the transfection conditions.
WO 39440 2017/048397
Table 4: BCL11A Gene Modification Levels by MiSeq Analysis
CD34+ Cell Transfection BCL11A Gene Modification (%)
Day 2 post Post-Thaw Day 14 of
TF differentiation
Prep #1 80 ug/mL 63014 + 20 ug/mL 78.6 81.4 72.0
65722
Untransfected 0. 1 0.2 0.2
Prep#2 80 ug/mL 63014 + 20 ug/mL 75.4 77.3 72.3
65722
Untransfected 0. 1 0. 1 0.0
These data show that CD34+ cell transfection with optimized pair
63014 and 65722 mRNA leads to very nt gene modification at the BCLl 1A
enhancer target site (>75% of alleles modified) and that the modification is
maintained very well (>90% retention of the modification) after freezing and thawing
of the cells and after oid differentiation.
The ZFN pair 63014/65722 was selected for further analysis. The
amino acid sequences for these ZFNs are shown below, where each comprises a
nuclear localization signal (NLS, Kalderon el al. (1984) Cell 39 (3 Pt 2):499-509) and
a hydrophilic peptide (Hopp el al. (1988) Nat Biotechnol 6: 1204-10) which enhances
on-target ZFN activity, both fused to the N—terrninal coding sequence. Thus, the
mRNA and amino acid sequences of the ZFNs are as follows:
63014 mRNA (1725 nt)
5 ’ gggagacaagcuuugaauuacaagcuugcuuguucuuuuugcagaagcucagaauaaacgcucaacuuugg
cagaucgaauucgccauggacuacaaagaccaugacggugauuauaaagaucaugacaucgauuacaaggaug
acgaugacaagauggcccccaagaagaagaggaaggucggcauccacgggguacccgccgcuauggcugagag
gcccuuccagugucgaaucugcaugcagaacuucagugaccaguccaaccugcgcgcccacauccgcacccaca
ccggcgagaagccuuuugccugugacauuugugggaggaaauuugcccgcaacuucucccugaccaugcauac
2O caagauacacacgggcagccaaaagcccuuccagugucgaaucugcaugcagaacuucaguuccaccggcaacc
ugaccaaccacauccgcacccacaccggcgagaagccuuuugccugugacauuugugggaggaaauuugccac
cuccggcucccugacccgccauaccaagauacacacgcacccgcgcgccccgaucccgaagcccuuccaguguc
gcaugcagaacuucagugaccaguccaaccugcgcgcccacauccgcacccacaccggcgagaagccu
uuugccugugacauuugugggaggaaauuugccgcccaguguugucuguuccaccauaccaagauacaccugc
ggggauccaucagcagagccagaccacugaacccgcacccggagcuggaggagaagaaguccgagcugcggcac
aagcugaaguacgugccccacgaguacaucgagcugaucgagaucgccaggaacagcacccaggaccgcauccu
ggagaugaaggugauggaguucuucaugaagguguacggcuacaggggaaagcaccugggcggaagcagaaa
gccugacggcgccaucuauacagugggcagccccaucgauuacggcgugaucguggacacaaaggccuacagc
ggcggcuacaaucugccuaucggccaggccgacgagauggagagauacguggaggagaaccagacccgggaua
agcaccucaaccccaacgagugguggaagguguacccuagcagcgugaccgaguucaaguuccuguucgugag
cggccacuucaagggcaacuacaaggcccagcugaccaggcugaaccacaucaccaacugcaauggcgccgugc
ugagcguggaggagcugcugaucggcggcgagaugaucaaagccggcacccugacacuggaggaggugcggc
gcaaguucaacaacggcgagaucaacuucagaucuugauaacucgagucuagaagcucgcuuucuugcugucc
uauuaaagguuccuuuguucccuaaguccaacuacuaaacugggggauauuaugaagggccuugagc
aucuggauucugccuaauaaaaaacauuuauuuucauugcugcgcuagaagcucgcuuucuugcuguccaauu
ucuauuaaagguuccuuuguucccuaaguccaacuacuaaacugggggauauuaugaagggccuugagcaucu
ggauucugccuaauaaaaaacauuuauuuucauugcugcgggacauucuuaauuaaaaaaaaaaaaaaaaaaaa
aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaacuag
(SEQ ID NO:28).
63014 amino acid sequence (recognition helix regions are underlined;
linker is shown in upper case italics; mutations to fingers I; 3 and 5 backbone residues
are shown in double-underlining; the dimerization domain mutations (ELD) are
shown in bold and italics; hilic peptide is indicated in lower case text; and the
nuclear localization signal (NLS) is shown in lowercase italics):
MdykdhdgdykdhdidykddddkMApkkkrkalHGVPAAMAERPFQCRICMQNF8w
NLRAHIRTHTGEKPFACDICGRKFARNFSLTMHTKIHTGSQKPFQCRICMQNF
SSTGNLTNHIRTHTGEKPFACDICGRKFATSGSLTRHTKIHTHPRAPIPKPFQCR
ICMQNFSDS 2SNLRAHIRTHTGEKPFACDICGRKFAAS HTKIHLRGSISR
ARPLNPHPELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMK
VYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEME
RYVEENQTRDKHLNPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNH
ITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS (SEQ ID
NO:29).
65722 mRNA (1680 nucleotides):
’ gggagacaagcuugaauacaagcuugcuuguucuuuuugcagaagcucagaauaaacgcucaacuuuggca
auucgccuagagaucuggcggcggagagggcagaggaagucuucuaaccugcggugacguggagga
gaaucccggcccuaggaccauggacuacaaagaccaugacggugauuauaaagaucaugacaucgauuacaagg
augacgaugacaagauggcccccaagaagaagaggaaggucggcauucaugggguacccgccgcuauggcuga
gaggcccuuccagugucgaaucugcaugcagaaguuugcccgcaacgaccaccgcaccacccauaccaagauac
acacgggcgagaagcccuuccagugucgaaucugcaugcagaacuucagucagaaggcccaccugauccgccac
auccgcacccacaccggcgagaagccuuuugccugugacauuugugggaggaaauuugcccagaagggcaccc
ugggcgagcauaccaagauacacacgggaucucagaagcccuuccagugucgaaucugcaugcagaacuucag
ucgcggccgcgaccugucccgccacauccgcacccacaccggcgagaagccuuuugccugugacauuuguggg
aggaaauuugcccgccgcgacaaccugcacucccauaccaagauacaccugcggggaucccagcuggugaagag
cgagcuggaggagaagaaguccgagcugcggcacaagcugaaguacgugccccacgaguacaucgagcugauc
gccaggaacagcacccaggaccgcauccuggagaugaaggugauggaguucuucaugaagguguacg
gcuacaggggaaagcaccugggcggaagcagaaagccugacggcgccaucuauacagugggcagccccaucga
cgugaucguggacacaaaggccuacagcggcggcuacaaucugccuaucggccaggccgacgagaug
cagagauacgugaaggagaaccagacccggaauaagcacaucaaccccaacgagugguggaagguguacccuag
cagcgugaccgaguucaaguuccuguucgugagcggccacuucagcggcaacuacaaggcccagcugaccagg
cugaaccgcaaaaccaacugcaauggcgccgugcugagcguggaggagcugcugaucggcggcgagaugauca
aagccggcacccugacacuggaggaggugcggcgcaaguucaacaacggcgagaucaacuucugauaacucga
gucuagaagcucgcuuucuugcuguccaauuucuauuaaagguuccuuuguucccuaaguccaacuacuaaac
ugggggauauuaugaagggccuugagcaucuggauucugccuaauaaaaaacauuuauuuucauugcugcgc
uagaagcucgcuuucuugcuguccaauuucuauuaaagguuccuuuguucccuaaguccaacuacuaaacugg
gggauauuaugaagggccuugagcaucuggauucugccuaauaaaaaacauuuauuuucauugcugcgggac
auucuuaauuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaacuag
(SEQ ID N030).
65722 amino acid sequence (recognition helix regions are underlined;
2O linker is shown in upper case italics; hydrophilic peptide is in lower case; nuclease
localization signal is in lower case italics; mutations to fingers I; 2 and 4 backbone
residues are shown in -underlining; the dimerization domain mutations (ELD)
are shown in bold and italics; and the FokI phosphate contact mutation(s) is shown in
wavy underlining):
MdykdhdgdykdhdidykddddkMApkkkrkalHGVPAAMAERPFQCRICMQKFAM
mHTKIHTGEKPFQCRICMQNFSS RHIRTHTGEKPFACDICGRKFAQ
KGTLGEHTKIHTGSQKPFQCRICMQNFSRGRDLSRHIRTHTGEKPFACDICGR
KFARRDNLHSHTKIHLRGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNST
QDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKA
YSGGYNLPIGQADEMQRYVKENQTRNKHINPNEWWKVYPSSVTEFKFLFVS
GPlFfiGNYKAQLTRLNRKTNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFN
NGEINF (SEQ ID N03 1).
Example 5: Assessment of Globin levels in Erythroid Progeny
Levels of OL— and y-
, B- globin mRNA in cellular mRNA ed at
day 14 of the entiation (before overall mRNA levels decline ically in the
course of enucleation and erythroid tion) were determined by R for the
two cell preps shown above in Table 4. The y- globin mRNA values are shown
normalized relative to the B- globin mRNA (Figure 5A) or to the OL— globin mRNA
values (Figure SE) from the same samples (using arbitrary units based on the ratio in
the untransfected RT-PCR rd defined as 1).
Reverse phase HPLC of protein s isolated at day 21, the end
point of the erythroid differentiation, was used to determine whether ZFN mediated
modification of the BCL11A erythroid enhancer elevates fetal hemoglobin at the
protein level. The Gamma globin (sum of the Agamma and Ggamma peaks) to alpha
globin ratios were determined, as well as the Gamma globin (sum of the Agamma and
Ggamma peaks)/ over beta-like globin ratios (sum of the Agamma, Ggamma, beta and
delta- globin peaks) and are shown in Figure 6.
In this ment an approximate 3-4 fold elevation of fetal globin
protein percentages to levels of about 15%-20% was observed in erythroid progeny of
HSPCs upon 63014/65722-mediated disruption of the BCL11A enhancer.
2O Example 6: Engraftment of edited cells in NSG mice
Edited human CD34+ cells were then injected into NSG mice to assess
engraftment. The extent of human chimerism (zle. the percentage of human CD45+
cells) was measured using cence activated cell sorting (FACS) in peripheral
blood collected at 8, and 12, 16 and 20 weeks post transplantation, and in bone
marrow collected at 12 weeks and 20 weeks. In addition, to test the level of
engraftment of ZFN modified cells, the level of gene disruption at the BCL11A
enhancer locus was evaluated by direct high-throughput sequencing of the ZFN target
locus and compared to the levels of target gene modification measured in the input
material.
[0226] HSPC from two y donors (termed PB-MR-OO3 and PB-MR-OO4)
were mobilized with G—SCF and Plerixafor and purified as described in Yannaki el al.
((2012) M0] Ther 20(1):230-8. doi: 10.1038/mt2011). Platelet depletion was
performed on the leukapheresis product using the Fresenius-Kabi Lovo device before
WO 39440
it was enriched for CD34+ cells using the Miltenyi Biotech CliniMACS Plus
ment. The d cells were then seeded in culture for transfection.
Two days after CD34+ cell purification, the cells were electroporated
using the Maxcyte instrument in the presence of either 120 ug/mL of a single mRNA
encoding the parental ZFN pair, 63014/65722 or optimized amounts of two separate
mRNAs encoding the optimized ZFN pair 80 ug/mL 63014 and 20 ug/mL 65722.
Before transfection, an aliquot of the cells were set aside as the untransfected l.
95 million cells were transfected from PB-MR—OO3 and 120 million cells were
transfected from PB-MR-OO4.
[0228] Following electroporation, a transient overnight e at 30°C was
performed and cells were then cultured for an additional 24 hours at 37°C. Two days
post-electroporation, cells aliquots for DNA analysis were taken and the remaining
cells were harvested, cryopreserved, and stored in liquid nitrogen.
Conditioning: Mice were treated with 10 mg/kg/day Baytril water 1-2
days prior to irradiation and sublethally irradiated with 300 RAD 16-24 hours before
transplantation. Transplantation was performed via tail vein injection (see below).
Then mice received fresh l water. l water was replace one week later and
Baytril water addition was discontinued 14 days after transplantation.
Transplantation: On the day of transplantation, m X-Vivo 10/
2O 1%PSG + 3 cytokine cocktail (Recombinant Human Stem Cell Factor (SCF),
Recombinant Human Thrombopoietin (TPO), and inant Human Flt-3 Ligand
(Flt-3L)) at 37°C, prepare fresh PB S/O. 1% BSA at ambient temperature
(sterile/filtered). The cryopreserved cells were thawed at 37°C, pelleted, resuspended
in pre-warmed X-Vivo medium, pelleted again, resuspended in PB S/O. 1% BSA and
counted. After another pelleting the cell pellet was resuspended in 550 uL per mouse
of PBS/O. 1%B SA (2 X 10/‘6 cells/mL based off the cell count). Cells were then
injected at room temperature into mouse tail vein with a 25 gauge needle. Study
groups are shown below in Table 5.
Table 5: Dosing groups for Engraftment of edited hCD34+ cells
Species: Mouse Sex, Age, : 60 Female NSG Mice
Grou N / N / Sacrifice
. % indels Viability Dose
Test Article
p No. Group (Day 2) 1d (cells/mous
Week 20
Post-Thaw
PB-MR-OO3 donor
cells treated with
l ’ 79%)o 83A)o 1 million 5
(Ml-10) 63014and 65722
mRNA.
PB-MR- 5
3 0'11)o 95%)0 1 Ion 5
(M21-30) 003.untransfected
PB-MR-OO4 donor cells
4 treated with 63014 75% 77% 1 n
( M3140)
and 65722 mRNA.
PB-MR- 5
6 (M5110-50) 0'11)0 92%)0 1 ml ||Ion
004. untransfected
Animals were ed daily for general health and weighed daily for
the first 2 weeks and weighed kly thereafter. Peripheral blood was collected
from the submandibular vein (100 uL) at 8, 12, 16 and 20 weeks post transplantation
or via cardiac puncture (1 mL) for the sacrificed animals at 12 and 20 weeks
post-transplantation. Half of the animals in each groups (5 mice per group) were
euthanized at 12 weeks post transplantation and bone marrow and terminal blood
were collected for analysis. The remaining animals in each group (5 mice per group)
were sacrificed at 20 weeks post transplantation.
Blood collection, cell harvest and processing: Peripheral blood was
collected via the submandibular vein or cardiac puncture into EDTA tubes and
centrifuged at 500 X g for 5 min to remove the plasma. Following phosphate-buffered
saline (PBS) bovine serum n (B SA) wash and centrifugation, a 10X volume of
hemolytic buffer was added to the pellet, and the mixture was incubated at 37°C for
min, centrifuged and washed again. The pelleted fraction was reconstituted in
1 mL PBS BSA, an aliquot was removed and fuged at 1,000 X g for 5 min, with
the resultant pellet preserved for genotyping. The supernatant fraction was utilized
for FACS analyses.
[0233] Bone marrow, femur, tibia and pelvic bones were collected in Iscove’s
Modified Dulbecco’s Medium (IMDM) ning fetal calf serum (FCS), total bone
marrow was flushed into a PBS BSA solution and filtered using a 70 um nylon
strainer. Volume was adjusted to 10 mL with PBS BSA, and an t was used for
cell counting (Cellometer).
ZFN activity was analyzed using MiSeq deep sequencing. In brief,
Genomic DNA from mice injected with either sfected control CD34+ HSPC or
CD34+ HSPC transfected with enhancer targeting ZFN mRNA was isolated from
blood samples obtained at 8 week and 12 week or from bone marrow at 12 weeks
post-injection. The region of interest (containing the ZFN binding site within the
BCL11A locus) was PCR amplified and the level of modification was determined by
paired end deep cing on the Illumina rm (MiSeq).
To generate libraries compatible with the Illumina MiSeq cing
platform, adaptors, barcodes, and flow cell binder (short DNA ce) were
ed to the target specific ons using two sets of fusion primers in sequential
PCRs. For MiSeq evaluations of human BCL11A enhancer modification in the mouse
blood and bone marrow samples, the protocol had to be adjusted due to the low target
DNA amounts in these samples.
The following primers were used for the MiSeq Adaptor PCR:
PRJIYLFN-f2:
ACA CGA CGC TCT TCC GAT CTN NNN AGT CCT CTT CTA CCC CAC CCA
(SEQ ID NO:32) and
PRJIYLFN-r4:
GAC GTG TGC TCT TCC GAT CTC TAC TCT TAG ACA TAA CAC ACC AGG
G (SEQ ID NO:33).
For the is, DNA from mouse bone marrow samples was isolated
by DNeasy and approximately 100 ng ofDNA were used in each PCR reaction. DNA
from mouse blood samples was isolated by Tissue XS and 10 uL of the 15 uL isolated
DNA was used in each reaction. In addition to the DNA, the following were added to
each MiSeq PCR reaction: 25 uL HotStar Taq miX (Qiagen), 0.5 uL each of the
BCL11A enhancer primers listed above (at a concentration of 100 nM), and water to a
50 uL total reaction . Typical MiSeq PCR conditions were: 95°C denaturation
for 15’, and 30 cycles at 94°C for 30”, 62°C for 30” and 72°C for 40”, followed by a
’ elongation at 72°C. After the MiSeq PCR, the PCR product was diluted between
1:50 and 1:200 with water, or left undiluted for samples with very low starting cell
numbers. Barcode PCR was performed with 1 uL of the MiSeq PCR product diluted
as described above, 25 uL HotStar Taq miX, 1 uL forward barcode primer, 1 uL
reverse barcode primer (both at a concentration of 10 nM) and water to a 50 uL total
reaction . Barcode PCR conditions were: 95°C denaturation for 15’, and 18
cycles at 94°C for 30”, 60°C for 30” and 72°C for 30”, ed by a 10’ elongation at
72°C. Barcode PCR products were pooled and sequenced on the Illumina MiSeq
sequencer. The results are shown in Table 5 above.
FACS analysis for chimerism, and cell lineage determination. To
assess the degree of human chimerism, the fraction of cells in the peripheral blood (at
8, l2, l6 and 20 weeks post engraftment) and bone marrow (at 12 and 20 weeks post
engraftment) were stained with hCD45-APC Cy7 (Biolegend) and hCD45-BV510
(BD Biosciences) antibodies respectively and FACS analysis was performed In
on, hematopoietic lineages analysis was performed by staining bone marrow
cells with the specific antibodies described in Table 6 below:
Table 6: Antibody sources for cell markers
TC: BD 561807 (clone UCHT1) BD 561807
CD19-PE: BD 340364 (clone SJZSCl) BD 340364
V510 BD 563204
Lin-APC (CD3/UCHT1, CD14/HCD14, CD16/3G8, CD19/HIBIQ, CD20/2H7, BIOLEGEND
BD 562492
DAKO 0870
BB 551400
BD 555398
BD 560710
BD 563671
BD 564232
BD 341051
BD 344612
BD 318310
BD 314520
BD 563780
In on, to purify and sort the HSPC populations, we used an
enrichment/depletion strategy using magnetic cell separation (MAC S). Bone marrow
cells were first stained with CDl9-biotin, CD3-biotin, B220-biotin, TERl l9-biotin
and m-ckit-biotin (BD ences) and then incubated with anti-biotin beads
(Miltenyi Biotec). The positive fraction and depleted fraction were separated using LS
columns (Miltenyi Biotec) placed in the magnetic field of a MACS. After tion,
the positive on was stained with Streptavidin-APC, CD3-FITC, CDl9-PE,
CD45-BV510 (BD ences) and the depleted fraction with CD34-FITC (BD
Biosciences), Gly-A-PE (DAKO), CDl9-APC (BD), Lin-APC (Biolegend),
Streptavidin-APC, CD45-BV510, CD33-PE-CF594 (BD) and CD3 8-PECy-7
(Biolegend).
Untransfected HSPC and 63014/65722 -transfected HSPC were
engrafted into NSG mice using standard procedures as described above. The degree
of human chimerism in these mice following engraftment was assessed by measuring
the fraction of hCD45 positive cells using FACS.
Figure 7 shows the percentages of human CD45+ cells in peripheral
blood collected at 8, and 12 weeks post-transplant and Figure 8 shows tages in
bone marrow harvested at Week 12. As shown, engraftment levels in this study were
comparable human chimerism following engraftment of sfected control and
63014/65722 transfected HSPC. Only 3 mice out of 60 distributed through the groups
did not have CD45+ cells indicating a failure to engraft.
Reconstitution of various hematopoietic cell es was tested by
FACS analysis of bone marrow cells obtained at week 12 with antibodies recognizing
lineage c cell surface s using standard procedures. As shown in
Figure 9, comparable representation of all analyzed human poietic lineages in
the bone marrow at week 12 post-injection between the BCLl 1A specific ZFN
ng mRNA treated CD34+ cell progeny and that of the untransfected cells was
observed. Bone marrow of the mice sacrificed at Week 12 post-engraftment was
2O isolated and the distribution of various hematopoietic lineages was analyzed by FACS
using dies recognizing the indicated lineage markers. All numbers are given as
the ratio of the cells staining positive for the indicated e marker versus the
percentage of human CD45 positive cells, except for the cells expressing the erythroid
marker Cd71+ (Ter119) in Figure 9C, which are given as the tage of positively
staining cells in the entire population since erythroid cells are not CD45 positive.
The levels of gene modification at the BCLl 1A erythroid enhancer (%
of alleles with insertions and deletions [indels]) were assessed by deep sequencing of
the ZFN target region using the MiSeq sequencing platform as described above. The
data are shown in Figure 10 for blood samples from week 8 and week 12, and in
Figure 11 for bone marrow samples from week 12 and sorted lineages derived from
the week 12 bone marrow cell samples of the 63014/65722 treated cells. For
comparison, the indel percentages measured 2 days after the transfection (as listed in
Table 5) are also shown on the graphs of Figures 10 and 11.
In addition, good retention of gene modification at the BCLl lA
erythroid enhancer was found for both 63014/65722 -treated HSPC donor sets at the
various time points and in the various lineages. Comparable modification was
observed in both BCLl lA dependent (B cells, ‘CDl9’, primitive progenitors,
‘CD3 8H’) and BCLl lA independent (myeloid ‘CD33’) lineages. Although the input
gene modification levels were higher in the PB-MR-OO3 donor sample than in the PB-
MR-OO4 donor sample, the PB-MR-OO4 derived cells consistently show higher
modification , 1'.e. better retention of modification, in mice than those derived
from PB-MR-OO3.
[0245] Overall, the observed retention of gene modification at the BCLl lA
erythroid enhancer in mice was consistent with that observed in prior mouse
experiments using a number of ZFNs targeting a y of gene s.
Furthermore, as human oid progenitors are not able to
entiate in mice, to determine the amount of BCLl lA ed gene modification
that occurred in these cells, bone marrow cells were removed from the mice and
differentiated in vilro. In these ments, bone marrow derived human cells were
removed from sacrificed mice at week 12 following engraftment and differentiated in
vitro as described above. BCLl lA target gene modification was ed by high-
throughput Miseq sequencing ofDNA isolated from cells at day 14 of the
differentiation.
Modification data (indels) are presented in Figure 12, which shows the
modification levels at day 14 of the erythroid differentiation. Indel percentages at day
14 of the in vitro differentiation vary markedly for each culture that was generated
from cells isolated from one mouse, ing the oligocellular nature of the
expansion obtained under these conditions. The data te that BCLl lA enhancer
modification mediated by the 63014/65722 ZFNs was not markedly altered during the
erythroid differentiation. As was observed in the blood and bone marrow samples,
erythroid progeny samples of PB-MR—OO4 derived cells showed higher average levels
of modification than erythroid progeny of OO3 derived cells.
[0248] The relative levels of various globin mRNAs were determined by RT-
PCR analysis ofRNA isolated from cells at day 14 of the in vitro erythroid
differentiation, and the data is presented in Figure 13A where the relative y-globin to
B globin mRNA and y-globin to in mRNA ratios (Figure 13B) averaged out for
the 5 erythroid cultures from each group. Both in the untransfected and the
63014/65722 treated samples y-globin to B globin or v-globin to 0L globin mRNA
ratios differ widely between the erythroid progenies of individual mice from the same
group. The donor PB-MR-OO4 derived cultures show on average lower v-globin ratios
than those from donor OO3, in line with the better tion observed for
PB-MR-OO4 derived samples. However in spite of this variability, the ZFN treated
sample averages show an ~1.5-2 fold increase in v globin mRNA levels compared to
their respective untransfected counterparts.
Globin n levels were assessed by HPLC analysis. Figure 14
shows globin protein analyses of samples harvested at day 16 of the differentiation.
The Gamma globin (sum of the Agamma and Ggamma peaks) to alpha globin ratios
were ined, as well as the Gamma globin (sum of the Agamma and Ggamma
peaks)/ over beta-like globin ratios (sum of the Agamma, Ggamma, beta and delta-
globin peaks) and the averages for each group are shown above each bar. In line with
the poor erythroid differentiation of the PB-MR—OO3 derived samples the gamma-
globin levels in the untransfected cells derived from this donor were very high
(~3 0%), and therefore ZFN treatment resulted in only a 1.2 fold increase in gamma-
globin levels. The PB-MR—OO4 showed more typical untransfected levels (~9%) and
exhibited an ~2-fold increase in gamma-globin protein levels after 12 weeks passage
through the mouse.
2O [0250] It is thought patients that have a >8.6% of v globin naturally are at an
age as compared to patients with v globin levels <8.6% (Platt el al. (1994) N
Engl JMed, 330: 163 9-44). In fact, achieving a chimeric 10-20% percentage of non-
sickle cell RBCs through engraftment of edited cells may lead to clinical
improvement (Chang el al. (2017) M0] Ther Methods Clin Dev 4: 137-148. doi
10. 1016/j .omtm.2016. 12.009). Thus, despite having to go through an in vitro
oid differentiation process, the percentage of chimeric cells, and the level of y-
globin protein being detected are indicative of therapeutic cy.
All patents, patent ations and publications ned herein are
hereby incorporated by reference in their entirety.
gh 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 ing from the spirit or scope of the disclosure. Accordingly,
the foregoing descriptions and examples should not be construed as limiting.
Claims (25)
1. l. A zinc finger nuclease comprising the amino acid sequence as shown in SEQ ID NO:29 or SEQ ID NO:3l.
2. A polynucleotide encoding one or more zinc finger nucleases according to claim l.
3. The polynucleotide of claim 2, wherein the polynucleotide is mRNA.
4. The polynucleotide of claim 3 comprising SEQ ID NO:28 or SEQ ID NO:30.
5. A cell comprising the zinc finger nucleases of claim 1 or the polynucleotide of any of claims 2 to 4.
6. The cell of claim 5, wherein the cell is a stem cell or precursor cell.
7. The cell of claim 6, wherein the cell is a human cell.
8. The cell of any of claims 5 to 7, wherein the genome of the cell is modified by the zinc finger nucleases.
9. The cell of claim 8, wherein the genomic modification is ed from the group consisting of insertions, deletions and combinations f.
10. The cell of claim 8 or 9, wherein the genomic modification is within the 30 +58 region of a BCLl lA er sequence.
11. A cell or cell line produced from the cell of any of claims 5 to 10.
12. A partially or fully differentiated cell descended from the cell or cell line of any of claims 5 to 11.
13. The cell of any of claims 5 to 12, wherein the cell exhibits increased expression of gamma and/or beta globin as compared to a cell without the genomic modification.
14. A pharmaceutical ition comprising the zinc finger nucleases 10 according to claim 1, the polynucleotide of any of claims 2 to 4 or the cell of any of claims 5 to 13.
15. A method of modifying an endogenous BCL11A enhancer sequence in a cell, the method comprising administering the zinc finger nucleases according to 15 claim 1 or the polynucleotide of any of claims 2 to 4 to the cell such that the nous BCL11A enhancer sequence is modified.
16. The method of claim 15, r comprising introducing an exogenous sequence into the cell such that the ous sequence is inserted into the 2O endogenous BCL11A enhancer sequence.
17. The method of claim 15, wherein the modification comprises a deletion.
18. A method of increasing globin production in a subject, the method 25 comprising: administering the cell of any of claims 5 to 13 to the subject.
19. The method of claim 18, wherein the subject is a human and the cell is a human stem cell or human sor cell.
20. The method of claim 19, wherein the cell is infused into the patient and the cell engrafts, differentiates and matures in the subject.
21. The method of any of claims 18 to 20, wherein the subject has a hemoglobinopathy.
22. The method of claim 21, wherein the hemoglobinopathy is a beta- thalassemia or sickle cell disease.
23. A method of producing a genetically modified cell comprising a genomic modification within an endogenous BCLl 1A enhancer sequence, the method comprising the steps of: a) ting a cell with the polynucleotide of any of claims 2 to 4, 10 b) subjecting the cell to conditions conducive to expressing the fusion protein from the polynucleotide, and c) modifying the endogenous BCLl 1A enhancer sequence with the expressed fusion n sufficient to produce the genetically modified cell. 15
24. The method of claim 23, further comprising stimulating the cell with at least one cytokine.
25. A kit comprising the zinc finger nuclease of claim 1, the polynucleotide of any of claims 2 to 4 and/or the cell of any of claims 5 to 13. 1 /2 1 CLEAVE GENOME INTEGRATE DONOR DOUBLE DONOR STRAND BREAK GENOME DONOR CE GENOME ADJACENT TO DONOR ADAPTOR I I 4 I I SEQUENCE REVEALS CANDIDATE CLEAVAGE SITE SUBSTITUTE SHEET (RULE 26)
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US62/378,978 | 2016-08-24 | ||
US62/443,981 | 2017-01-09 | ||
US62/545,778 | 2017-08-15 |
Publications (1)
Publication Number | Publication Date |
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NZ791706A true NZ791706A (en) | 2022-08-26 |
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