Note: Descriptions are shown in the official language in which they were submitted.
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REPLACEMENT OF RAG1 FOR USE IN THERAPY
FIELD OF THE INVENTION
The present invention relates to methods for gene-editing cells to introduce a
RAG1
polypeptide, for example as a treatment for severe combined immunodeficiency.
The present
invention also relates to polynucleotides, vectors, guide RNAs, kits,
compositions, and gene
editing systems for use in said methods. The present invention also relates to
genomes and
cells obtained or obtainable by said methods.
BACKGROUND TO THE INVENTION
The RAG1 and RAG2 proteins initiate V(D)J recombination, allowing generation
of a diverse
repertoire of T and B cells (Teng G, Schatz DG. Advances in Immunology.
2015;128:1-39).
RAG mutations in humans cause a broad spectrum of phenotypes, including T- B-
SCID,
Omenn syndrome (OS), atypical SCID (AS) and combined immunodeficiency with
granuloma/autoimmunity (CID-G/A1) (Notarangelo LD, et al. Nat Rev I mmunol.
2016; 16 (4):234-246) .
Hematopoietic stem cell transplantation (HSCT) is the mainstay for severe
forms of RAG1
deficiency, including T- B- SCID, OS and AS with an overall survival of ¨80%
after
transplantation from donors other than matched siblings (Haddad E, et al.
Blood.
2018;132(17):1737-49). However, overall survival rate is lower in non-matched-
sibling donors
and a high rate of graft failure and poor T and B cell immune reconstitution
are observed in
the absence of myeloablative or reduced intensity conditioning. Besides donor
type and
conditioning, other factors associated with worse outcomes after HSCT include
age (>3.5
months of life) and infections at the time of transplantation.
An alternative approach to overcome the obstacles with HSCT is represented by
gene therapy.
Selective advantage of gene-corrected hematopoietic stem cells (HSCs) to
overcome the
block of T and B cells that occur in the absence of RAG activity represents
the rationale for
developing such a strategy. In recent years, lentiviral vectors have become
the strategy of
choice to deliver the transgene of interest, and allow its expression under
the control of
suitable promoters (Naldini L, Nature. 2015;526:351-360). In the case of RAG1
deficiency, the
observation that endogenous RAG1 gene expression is tightly regulated during
cell cycle and
during lymphoid development, may expose to the risk that ectopic or
dysregulated gene
expression could lead to immune dysregulation or leukemia (Lagresle-Peyrou C,
et al. Blood.
2006;107(1):63-72; Pike-Overzet K, et al. Leukemia. 2011;25(9):1471-83; and
Pike-Overzet
K, et al. Journal of Allergy and Clinical Immunology. 2014;134:242-243).
Several groups have
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examined the safety and efficacy of lentivirus-mediated gene therapy for RAG
deficiency in
preclinical models showing poor immune reconstitution or severe signs of
inflammation, with
cellular infiltrates in the skin, lung, liver, kidney, and presence of
circulating anti-double strand
DNA (van Til NP, et al. J Allergy Clin Immunol. 2014;133(4):1116-23).
Overall, these data raise significant concerns on the clinical use of
conventional RAG1 gene
therapy vectors that allow suboptimal levels and deregulated pattern of gene
expression.
Thus, there is a demand for improved treatments for RAG1 deficiency.
SUMMARY OF THE INVENTION
The present inventors have developed a gene editing strategy to correct
mutations in the
RAG1 gene by targeting the genomic region located at the 5' of the second
exon, which
contains the entire coding sequence of the gene.
The present inventors have designed and selected a panel of CRISPR-Cas9
nucleases and
identified specific sites in non-repeated regions of the first intron of the
human RAG1 gene.
The present inventors have identified guide RNAs and optimal conditions for
the delivery of
the CRISPR-Cas9 nuclease ribonucleoprotein complexes. In parallel, the present
inventors
have developed a donor DNA carrying the human RAG1 cDNA.
The gene editing strategy allows a high level of activity (measured as
frequency of NHEJ-
mutagenesis) and targeting efficiency (measured as GFP expression), both in a
surrogate cell
line deficient in RAG1 expression and expressing a recombination cassette, and
in humans
CD34+ HSCs obtained from mobilized peripheral blood (mPB). High editing
efficiencies were
reached in mobilized peripheral blood (nnPB) CD34+ cells using the gene
editing strategy.
In one aspect, the present invention provides a polynucleotide comprising from
5' to 3': a first
homology region, a splice acceptor sequence, a nucleotide sequence encoding a
RAG1
polypeptide, and a second homology region.
In another aspect, the present invention provides a polynucleotide comprising
from 5' to 3': a
first homology region, a nucleotide sequence encoding a RAG1 polypeptide, and
a second
homology region.
In some embodiments:
(i) the first homology region is homologous to a first region of the RAG1
intron 1 and
the second homology region is homologous to a second region of the RAG1 intron
1;
Or
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(ii) the first homology region is homologous to a first region of the RAG1
intron 1 or the
RAG1 exon 2 and the second homology region is homologous to a second region of
the RAG1 exon 2.
In some embodiments, the first homology region is homologous to a first region
of the RAG1
intron 1 and the second homology region is homologous to a second region of
the RAG1 intron
1.
In some embodiments, the first homology region is homologous to a first region
of the RAG1
intron 1 and the second homology region is homologous to a second region of
the RAG1 exon
2.
In some embodiments, the first homology region is homologous to a first region
of the RAG1
exon 2 and the second homology region is homologous to a second region of the
RAG1 exon
2.
In some embodiments:
(i) the first homology region is homologous to a region upstream of chr 11:
36569295
and the second homology region is homologous to a region downstream of chr 11:
36569298;
(ii) the first homology region is homologous to a region upstream of chr 11:
36573790
and the second homology region is homologous to a region downstream of chr 11:
36573793;
(iii) the first homology region is homologous to a region upstream of chr 11:
36573641
and the second homology region is homologous to a region downstream of chr 11:
36573644;
(iv) the first homology region is homologous to a region upstream of chr 11:
36573351
and the second homology region is homologous to a region downstream of chr 11:
36573354;
(v) the first homology region is homologous to a region upstream of chr 11:
36569080
and the second homology region is homologous to a region downstream of chr 11:
36569083;
(vi) the first homology region is homologous to a region upstream of chr 11:
36572472
and the second homology region is homologous to a region downstream of chr 11:
36572475;
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(vii) the first homology region is homologous to a region upstream of chr 11:
36571458
and the second homology region is homologous to a region downstream of chr 11:
36571461;
(viii) the first homology region is homologous to a region upstream of chr 11:
36571366
and the second homology region is homologous to a region downstream of chr 11:
36571369;
(ix) the first homology region is homologous to a region upstream of chr 11:
36572859
and the second homology region is homologous to a region downstream of chr 11:
36572862;
(x) the first homology region is homologous to a region upstream of chr 11:
36571457
and the second homology region is homologous to a region downstream of chr 11:
36571460;
(xi) the first homology region is homologous to a region upstream of chr 11:
36569351
and the second homology region is homologous to a region downstream of chr 11:
36569354; or
(xii) the first homology region is homologous to a region upstream of chr 11:
36572375
and the second homology region is homologous to a region downstream of chr 11:
36572378.
In some embodiments:
(i) the first homology region is homologous to a region upstream of chr 11:
36569295
and the second homology region is homologous to a region downstream of chr 11:
36569298;
(ii) the first homology region is homologous to a region upstream of chr 11:
36573351
and the second homology region is homologous to a region downstream of chr 11:
36573354; or
(iii) the first homology region is homologous to a region upstream of chr 11:
36571366
and the second homology region is homologous to a region downstream of chr 11:
36571369.
In preferred embodiments, the first homology region is homologous to a region
upstream of
chr 11: 36569295 and the second homology region is homologous to a region
downstream of
chr 11: 36569298.
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In some embodiments, the first homology region is homologous to a region
upstream of chr
11: 36573790 and the second homology region is homologous to a region
downstream of chr
11:36573793.
In some embodiments, the first homology region is homologous to a region
upstream of chr
11: 36573641 and the second homology region is homologous to a region
downstream of chr
11:36573644.
In some embodiments, the first homology region is homologous to a region
upstream of chr
11: 36573351 and the second homology region is homologous to a region
downstream of chr
11: 36573354.
In some embodiments, the first homology region is homologous to a region
upstream of chr
11: 36569080 and the second homology region is homologous to a region
downstream of chr
11:36569083.
In some embodiments, the first homology region is homologous to a region
upstream of chr
11: 36572472 and the second homology region is homologous to a region
downstream of chr
11:36572475.
In some embodiments, the first homology region is homologous to a region
upstream of chr
11: 36571458 and the second homology region is homologous to a region
downstream of chr
11:36571461.
In some embodiments, the first homology region is homologous to a region
upstream of chr
11: 36571366 and the second homology region is homologous to a region
downstream of chr
11:36571369.
In some embodiments, the first homology region is homologous to a region
upstream of chr
11: 36572859 and the second homology region is homologous to a region
downstream of chr
11:36572862.
In some embodiments, the first homology region is homologous to a region
upstream of chr
11: 36571457 and the second homology region is homologous to a region
downstream of chr
11:36571460.
In some embodiments, the first homology region is homologous to a region
upstream of chr
11: 36569351 and the second homology region is homologous to a region
downstream of chr
11:36569354.
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In some embodiments, the first homology region is homologous to a region
upstream of chr
11: 36572375 and the second homology region is homologous to a region
downstream of chr
11:36572378.
In preferred embodiments, the first homology region is homologous to a region
comprising chr
11: 36569245-chr 11: 36569294 and/or the second homology region is homologous
to a region
comprising chr 11: 36569299-chr 11: 36569348.
In some embodiments, the 3' terminal sequence of the first homology region
comprises or
consists of a nucleotide sequence that has at least 70% identity to SEQ ID NO:
7 and/or the
5' terminal sequence of the second homology region comprises or consists of a
nucleotide
sequence that has at least 70% identity to SEQ ID NO: 19.
In some embodiments, the first homology region comprises or consists of a
nucleotide
sequence that has at least 70% identity to SEQ ID NO: 31, or a fragment
thereof and/or the
second homology region comprises or consists of a nucleotide sequence that has
at least 70%
identity to SEQ ID NO: 32, or a fragment thereof.
In some embodiments, the first and second homology regions are each 50-1000bp
in length,
100-500 bp in length, or 200-400 bp in length.
In some embodiments, the nucleotide sequence encoding a RAG1 polypeptide
comprises or
consists of a nucleotide sequence encoding an amino acid sequence that has at
least 70%
identity to SEQ ID NO: 4 or SEQ ID NO: 5.
In some embodiments, the nucleotide sequence encoding a RAG1 polypeptide
comprises or
consists of a nucleotide sequence that has at least 70% identity to SEQ ID NO:
6.
In some embodiments, the splice acceptor site comprises or consists of a
nucleotide sequence
that has at least 70% identity to SEQ ID NO: 33.
In preferred embodiments, the nucleotide sequence encoding a RAG1 polypeptide
is operably
linked to a polyadenylation sequence, optionally wherein the polyadenylation
sequence is a
bGH polyadenylation sequence.
In some embodiments, the nucleotide sequence encoding a RAG1 polypeptide is
operably
linked to a polyadenylation sequence comprising or consisting of a nucleotide
sequence that
has at least 70% identity to SEQ ID NO: 35.
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In some embodiments, the nucleotide sequence encoding a RAG1 polypeptide is
operably
linked a Kozak sequence, optionally wherein the Kozak sequence comprises or
consists of a
nucleotide sequence that has at least 70% identity to SEQ ID NO: 36.
In some embodiments, the polynucleotide comprises or consists of a nucleotide
sequence that
has at least 70% identity to SEQ ID NO: 39.
In another aspect, the present invention provides a vector comprising the
polynucleotide of
the invention.
In some embodiments, the vector is a viral vector, optionally an adeno-
associated viral (AAV)
vector such as an AAV6 vector. In some embodiments, the vector is a lentiviral
vector, such
as an integration-defective lentiviral vector (IDLV).
In another aspect, the present invention provides a guide RNA comprising or
consisting of a
nucleotide sequence that has at least 90% identity to any of SEQ ID NOs: 41-
52.
In another aspect, the present invention provides a guide RNA comprising or
consisting of a
nucleotide sequence that has at least 90% identity to any of SEQ ID NOs: 53-
55.
In preferred embodiments, the guide RNA comprises or consists of a nucleotide
sequence
that has at least 90% identity to SEQ ID NO: 41. In preferred embodiments, the
guide RNA
comprises or consists of a nucleotide sequence that has at least 90% identity
to SEQ ID NO:
53. In some embodiments, the guide RNA comprises or consists of a nucleotide
sequence
that has at least 90% identity to SEQ ID NO: 42. In some embodiments, the
guide RNA
comprises or consists of a nucleotide sequence that has at least 90% identity
to SEQ ID NO:
43. In some embodiments, the guide RNA comprises or consists of a nucleotide
sequence
that has at least 90% identity to SEQ ID NO: 44. In some embodiments, the
guide RNA
comprises or consists of a nucleotide sequence that has at least 90% identity
to SEQ ID NO:
45. In some embodiments, the guide RNA comprises or consists of a nucleotide
sequence
that has at least 90% identity to SEQ ID NO: 46. In some embodiments, the
guide RNA
comprises or consists of a nucleotide sequence that has at least 90% identity
to SEQ ID NO:
47. In some embodiments, the guide RNA comprises or consists of a nucleotide
sequence
that has at least 90% identity to SEQ ID NO: 48. In some embodiments, the
guide RNA
comprises or consists of a nucleotide sequence that has at least 90% identity
to SEQ ID NO:
49. In some embodiments, the guide RNA comprises or consists of a nucleotide
sequence
that has at least 90% identity to SEQ ID NO: 50. In some embodiments, the
guide RNA
comprises or consists of a nucleotide sequence that has at least 90% identity
to SEQ ID NO:
51. In some embodiments, the guide RNA comprises or consists of a nucleotide
sequence
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that has at least 90% identity to SEQ ID NO: 52. In some embodiments, the
guide RNA
comprises or consists of a nucleotide sequence that has at least 90% identity
to SEQ ID NO:
54. In some embodiments, the guide RNA comprises or consists of a nucleotide
sequence
that has at least 90% identity to SEQ ID NO: 55.
In some embodiments, from one to five of the terminal nucleotides at 5' end
and/or 3' end of
the guide RNA are chemically modified to enhance stability, optionally wherein
three terminal
nucleotides at 5' end and/or 3' end if the guide RNA are chemically modified
to enhance
stability, optionally wherein the chemical modification is modification with
2'-0-methyl
3'phosphorothioate.
In another aspect, the present invention provides a kit comprising the
polynucleotide or the
vector of the invention.
In another aspect, the present invention provides a composition comprising the
polynucleotide
or the vector of the invention.
In another aspect, the present invention provides a gene-editing system
comprising the
polynucleotide or the vector of the invention.
In some embodiments, the kit, composition, or gene-editing system further
comprises a guide
RNA of the invention. In some embodiments, the kit, composition, or gene-
editing system
further comprises a RNA-guided nuclease, optionally wherein the RNA-guided
nuclease is a
Cas9 endonuclease
In another aspect, the present invention provides for use of the
polynucleotide, the vector, the
kit, the composition, or the gene-editing system, for gene editing a cell or a
population of cells.
In some embodiments, the use is ex vivo or in vitro use.
In another aspect, the present invention provides a genome comprising the
polynucleotide of
the invention.
In another aspect, the present invention provides a genome comprising a splice
acceptor
sequence and a nucleotide sequence encoding a RAG1 polypeptide located in the
RAG1
intron 1 or RAG1 exon 2. In some embodiments, the splice acceptor sequence and
the
nucleotide sequence encoding RAG1 are located in the RAG1 intron 1.
In some embodiments:
(i) the splice acceptor sequence and the nucleotide sequence encoding RAG1
replace
chr 11: 36569295 to chr 11: 36569298;
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(ii) the splice acceptor sequence and the nucleotide sequence encoding RAG1
replace
chr 11: 36573790 to chr 11: 36573793;
(iii) the splice acceptor sequence and the nucleotide sequence encoding RAG1
replace chr 11: 36573641 to chr 11: 36573644;
(iv) the splice acceptor sequence and the nucleotide sequence encoding RAG1
replace chr 11: 36573351 to chr 11: 36573354;
(v) the splice acceptor sequence and the nucleotide sequence encoding RAG1
replace
chr 11: 36569080 to chr 11: 36569083;
(vi) the splice acceptor sequence and the nucleotide sequence encoding RAG1
replace chr 11: 36572472 to chr 11: 36572475;
(vii) the splice acceptor sequence and the nucleotide sequence encoding RAG1
replace chr 11: 36571458 to chr 11: 36571461;
(viii) the splice acceptor sequence and the nucleotide sequence encoding RAG1
replace chr 11:36571366 to chr 11:36571369;
(ix) the splice acceptor sequence and the nucleotide sequence encoding RAG1
replace chr 11: 36572859 to chr 11: 36572862;
(x) the splice acceptor sequence and the nucleotide sequence encoding RAG1
replace
chill: 36571457 to chr 11:36571460;
(xi) the splice acceptor sequence and the nucleotide sequence encoding RAG1
replace chr 11: 36569351 to chr 11: 36569354; or
(xii) the splice acceptor sequence and the nucleotide sequence encoding RAG1
replace chr 11: 36572375 to chr 11: 36572378.
In some embodiments:
(i) the splice acceptor sequence and the nucleotide sequence encoding RAG1
replace
chr 11: 36569295 to chr 11: 36569298;
(ii) the splice acceptor sequence and the nucleotide sequence encoding RAG1
replace
chr 11: 36573351 to chr 11: 36573354; or
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(iii) the splice acceptor sequence and the nucleotide sequence encoding RAG1
replace chr 11:36571366 to chr 11:36571369.
In some embodiments, the splice acceptor sequence and the nucleotide sequence
encoding
RAG1 replace chr 11: 36569295 to chr 11: 36569298.
In another aspect, the present invention provides a cell comprising the
polynucleotide, the
vector, or the genome of the invention.
In another aspect, the present invention provides a population of cells
comprising one or more
cells of the present invention.
In another aspect, the present invention provides a method of gene editing a
population of
cells comprising delivering the polynucleotide or the vector of the invention
to a population of
cells to obtain a population of gene-edited cells. In some embodiments, the
method is an ex
vivo or in vitro method.
In another aspect, the present invention provides a method of treating
immunodeficiency in a
subject in need thereof, comprising delivering the polynucleotide or the
vector of the invention
to a population of cells to obtain a population of gene-edited cells and
administering the
population of gene-edited cells to the subject.
In another aspect, the present invention provides a population of gene-edited
cells obtainable
by the method of the invention.
In another aspect, the present invention provides the polynucleotide, the
vector, the guide
RNA, the kit, the composition, or the gene-editing system, for use in treating
immunodeficiency
in a subject.
In another aspect, the present invention provides a method of treating a
subject comprising
administering a cell, a population of cells, or a population of gene edited
cells of the present
invention to the subject.
In another aspect, the present invention provides a method of treating
immunodeficiency in a
subject in need thereof comprising administering a cell, a population of
cells, or a population
of gene edited cells of the present invention to the subject.
In another aspect, the present invention provides a cell, a population of
cells, or a population
of gene edited cells of the present invention for use as a medicament.
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In another aspect, the present invention provides a cell, a population of
cells, or a population
of gene edited cells of the present invention for use in treating
immunodeficiency in a subject.
DESCRIPTION OF DRAWINGS
Figure 1. Generation of NALM6 Cas9 and K562 Cas9 cell lines
A) Schematic representation of the gene correction approach; B) Schematic
representation of
the protocol for generation of K562 Cas9 and NALM6 Cas9 cell lines; C) Vector
Copy Number
(VCN) of the integrated Cas9 containing cassette measured by ddPCR, telomerase
was used
as normalizer; D) Cas9 expression for scaling doses of doxycycline measured by
qPCR in
NALM6 Cas9 (left panel) and K562 Cas9 (right panel) cell lines, represented as
fold change
Vs actin.
Figure 2. Selection of the best performing gRNA
A) Schematic representation of the intronic and exonic loci targeted by the
different gRNA
tested; B) Schematic representation of the experimental protocol; C)
Percentages of NHEJ
induced indels in K562 Cas9 treated with different doses of plasmids encoding
for different
guides, 7 days after transfection, n=1; D) Percentages of NHEJ induced indels
in NALM6 Cas9
treated with different doses of plasmids encoding for guides 3, 7 and 9, 7
days after
transfection, n=1; E) Percentages of NHEJ induced indels in NALM6 Cas9 treated
with
different doses of guides 3 and 9 in vitro preassembled RNPs 7 days after
transfection, n=1.
Figure 3. Donor DNA optimization
A) RAG1 gene expression measured by RT-qPCR, represented as fold change vs
RAG1
expression in 293T cell line, actin was used as normalizer; B) Schematic
representation of
different SA GFP DNA donor tested; C) Schematic representation of the splicing
mechanism
with SA_GFP_SD donor; D) Percentage of targeted cells measured by flow
cytometry as
GFP+ cells, 7 days after transfection; E) GFP expression levels measured as
Mean
Fluorescence Intensity (MFI) gating on GFP+ events; F) Representative FlowJo
plots; One-
way ANOVA, Geisser-Greenhouse correction for multiple comparison, n=3. P
values: *<0.05;
**<0.005; ***<0.0005; ****<0.0001. Mean SD are shown.
Figure 4. Off-target analysis
A) Table shows the top 10 off-target sites predicted by in silico COSMID tool
for guide 9. The
off-target sequence, type of PAM, score, number of mismatches and chromosomal
position
are shown. B-C) Cutting efficiency measured as percentage of NHEJ (D) and
dsDNA tag
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integration (ODN) on target site are evaluated by RFLP in K562 cells. D-E)
Plots show the
coverage of on-target reads (chromosome 11) of guide 9 (D) and guide 7 (E) and
off-target
reads identified for guide 7 by relaxed constraints (chromosome 20 and 9).
Figure 5. Optimization of the gene editing protocol, guide 3 efficiency
A) Schematic representation of the gene editing protocol; B) Schematic
representation of the
gating strategy; C) Percentages of NHEJ induced indels in hCB-CD34+ cells
treated with
different doses of guides 3 and 9 as in vitro preassembled RNPs, n=2; D)
Percentage of
targeted cells using guide 3, measured by flow cytometry as GFP+ cells in the
hCD34+ gate,
n=1; E) Percentage of targeted cells using guide 3, measured by flow cytometry
as GFP+ cells
in the three main hCD34+ cell subpopulations for hCD133 hCD90 expression, n=1.
Figure 6. Optimization of the gene editing protocol, guide 9 efficiency
A) Percentages of viable cells measured by flow cytometry as 7AADIAnnexinV- at
day 4; B)
Total number of cells at day 7 expressed as fold increase compared day 3; C)
Frequency of
hCD34+ cells at day 7 measured by flow cytometry; D) Distribution of the 3
hCD34+ cell
subpopulations measured by flow cytometry based on the expression of hCD133
and hCD90
at day 7; E) Frequency of targeted cells measured by flow cytometry as GFP+
cells in the 3
hCD34+ cell subpopulations based on the expression of hCD133 and hCD90 at day
7; F)
Percentages of targeted cells measured by ddPCR at day 7, telomerase genomic
site was
used as normalizer; G) Total number of edited cells at day 7 calculated on
frequency of
targeted cells by ddPCR. One-way ANOVA, Geisser-Greenhouse correction for
multiple
comparison, n=3. P values: *<0.05; **<0.005; ***<0.0005; ****<0.0001. Mean SD
are shown.
Figure 7. In vivo transplantation of gene edited hCB-CD34+ cells
A) Percentages of targeted cells measured by ddPCR at day 4, telomerase
genomic site was
used as normalizer; B) Treated cell engraftment measured by flow cytometry as
frequency of
hCD45+ cells in peripheral blood (PB); C) Targeted cell engraftment measured
in PB by flow
cytometry as frequency of GFP+ cells in hCD45+ gate; D, F, H) B cell, T cell
and Myeloid cell
frequency in PB measured as percentage of hCD19+ cells (D), hCD3+ cells (F),
hCD13+ cells
(F) in hCD45+ gate, respectively. E, G, I) Targeted cells among the B-cell, T-
cell and Myeloid-
cell compartment in PB measured as GFP+ cells in the hCD19+ gate (E), hCD3+
gate (G) and
hCD13+ gate (I), respectively; L) Frequency of hCD34+ cells measured by flow
cytometry
among hCD45+ cells in the bone marrow; M) Frequency of targeted cells measured
by flow
cytometry as GFP+ cells among hCD34+ cells in the bone marrow; N) Frequency of
GFP+
expressing cells measured by flow cytometry, among different T-cell
development stages in
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the thymus (according to the expression of hCD4 and hCD8), in the peripheral
blood and in
the spleen (according to the expression of hCD3, hCD4 and hCD8), 17 weeks
after transplant.
Mann-Whitney test at 17 weeks after transplant. Group size: SA_GFP n=5;
PGK_GFP n=4. P
values: *<0.05; **<0.005; ***<0.0005; ****<0.0001. Mean SD are shown.
Figure 8. Test corrective donor on hMPB-CD34+ cells
A) Schematic representation of the corrective donor; B) Schematic
representation of the
experimental protocol; C) Percentages of targeted cells measured by ddPCR on
sorted
hCD34+ cell subpopulation according to the expression of hCD133 and hCD90 at
day 4,
telomerase genomic region was used as normalizer; D) Total number of cells at
day 4
represented as fold increase compared day 0. N=3.
Figure 9. In vivo transplantation of edited hMPB-CD34+ cells from HD and RAG1-
patient
A) Schematic representation of the experimental groups; B) Percentage of
targeted cells
measured by ddPCR at day 4, telomerase genomic region was used as normalizer;
C) Cell
engraftment measured by flow cytometry in PB as frequency of hCD45+ cells; D)
Frequency
of targeted cells among human cells measured by ddPCR in PB 8 weeks after
transplant,
telomerase genomic region was used as normalizer; E) Immune cell distribution
in PB of mice
transplanted with MPB-CD34+ of HD treated and untreated cells measured by flow
cytometry
according to the expression of hCD19, hCD3 and hCD13 in the hCD45+ gate; F)
Immune cell
distribution in PB of mice transplanted with MPB-CD34+ cells derived from a
RAG1-patient
treated and untreated cells measured by flow cytometry according to the
expression of hCD19,
hCD3 and hCD13 in the hCD45+ gate; G, H) Analyses in bone marrow (G) and
spleen (H) of
the proportion of human engraftment measured as frequency of hCD45+ cells by
flow
cytometry (left panels) and of targeting efficiency measured as HDR by ddPCR
(right panels).
Mean SD are shown.
Figure 10. Multiparametric analysis of hMPB-CD34+ cells from HD and RAG1-
patient
before and after gene editing manipulation.
A, B) Analysis of HSPC composition was performed in MPB-CD34+ cells derived
from healthy
donor (HD, A) and a RAG1-Patient (Pt, B) by flow-cytometry. The analysis was
performed
before the expansion phase (day-3) and 1 day after the gene editing procedure
(GE).
Untreated cells (UT) were also analyzed the same day of edited cells. Graphs
show 20
subtypes analyzed in the Lineage negative (Lin-) CD34+ gate including:
Hematopoietic Stem
cells (HSC), Multipotent Progenitors (MPP), Multi-Lymphoid Progenitors (MLP),
Early T
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Progenitors (ETP), B and NK cell precursors (Pre-B/NK), common myeloid
progenitors (CM F),
granulocyte-monocyte progenitors (GM P), megakaryoerythroid progenitors (MEP),
megakaryocyte progenitors (MKp) and erythroid progenitors (EP).
Figure 11. Donor Screening for RAG1 editing.
A) Schematic representations of donor constructs. HA_L, left homology arm;
HA_R, right
homology arm; SA, splice acceptor; SD, splice donor; BGHpA, bovine growth
hormone poly
A; WPRE, Woodchuck hepatitis virus post-transcriptional regulatory element;
IRES, the
internal ribosome entry site sequence; PEST, proline (P), glutamic acid (E),
serine (S), and
threonine (T). B) schematic representation of the experimental protocol. C)
GFP expression
levels shown as Mean Fluorescence Intensity (MFI) gating on GFP+ events
measured by flow
cytometry over time (d, days after editing). D) Modulation of GFP expression
in serum starved
cells is shown as ratio of GFP MFI of starved cells (- FBS) and GFP MFI of not
starved cells
(+ FBS) (1 experiment representative of 3).
Figure 12. Editing enhancer effects on HDR efficiency of RAG1 locus.
A) Schematic representation of the gene editing protocol (upper panel) and
artificial thymic
organoid protocol (ATO) (lower panel). B) HDR efficiency is shown as
percentages of edited
alleles measured by ddPCR 7 days after editing; C) Frequency of targeted cells
measured by
flow cytometry as GFP + cells among hCD34+ subsets 7 days after editing; D)
Analysis of
HSPC composition was performed in MPB or BM CD34+ cells derived from healthy
donor by
flow-cytometry. The analysis was performed before the expansion phase (day 0)
and 1 day
after the gene editing procedure (GE, day 4). Untreated cells (UT) were also
analyzed the
same day of edited cells. Graphs show 20 subtypes analyzed in the Lineage
negative (Lin-)
CD34+ gate including: Hematopoietic Stem cells (HSC), Multipotent Progenitors
(MPP), Multi-
Lymphoid Progenitors (MLP), Early T Progenitors (ETP), B and NK cell
precursors (Pre-B/NK),
common myeloid progenitors (CMP), granulocyte-monocyte progenitors (GMP),
megakaryo-
erythroid progenitors (MEP), megakaryocyte progenitors (MKp) and erythroid
progenitors
(EP).
Figure 13. Editing enhancer effects on T cell differentiation potential.
Representative images of artificial thymic organoid (ATO) 4 weeks after ATO
seeding with
Untreated cells (UT) or edited cells with or without HDR enhancers. B) total
number of cells
harvested from ATOs 4 weeks after ATO seeding. C) HDR efficiency is shown as
percentages
of edited alleles measured by ddPCR in bulk differentiated T cells 4 weeks
after ATO seeding.
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D) HDR efficiency is measured as percentage of GFP+ cells within distinct T
cell subpopulation
by flow cytometry 4 weeks after ATO seeding.
Figure 14. Donor constructs for the intronic correction strategy.
Schematic representation of the SA_coRAG1 CDS_BGHpA (A) and SA_coRAG1 CDS_SD
(B) donor templates used for the intronic correction strategy. HA, homology
arm; SA, splice
acceptor; SD, splice donor; coRAG1 CDS, codon optimized RAG1 coding sequence;
BGHpA,
bovine growth hormone poly A; Ex., exon; gRNA, guide RNA; 3'UTR, 3'
untranslated region;
HDR, homology directed repair
Figure 15. Corrective donor comparison in NALM6.Rag1K0 cells.
(A) Schematic representation of the experiment performed to compare the
correction efficacy
of the two donors: the SA_coRAG1 CDS_BGHpA vs the SA_coRAG1 CDS_SD donor. (B)
RAG1 CDS expression was evaluated in various NALM6.Rag1K0 edited clones by RT-
qPCR
and measured as relative expression to the housekeeping beta-actin. (C)
Recombination
activity was evaluated 7 days after serum-starvation as proportion of GFP+
cells gated on
transduced cells by flow cytometry.
Figure 16. Corrective donor comparison in HD-HSPC.
(A) Hematopoietic stem and progenitor cells were edited by guide 9 and Cas9 as
RNP in
combination with SA_coRAG1 CDS_BGHpA or SA_coRAG1 CDS_SD donor. The proportion
of edited alleles was evaluated by ddPCR in bulk HSPC 4 days after the
editing. (B) The
proportion of edited alleles was evaluated by ddPCR in HSPC subsets isolated
by cell sorting.
(C) Kinetics of cell growth in untreated (UT) or edited HSPC according to the
indicated donors,
doses and days after gene editing (GE). (D) Colony forming unit (CFU) assay
was performed
on untreated or edited HSPC by counting the number of red (erythroid), white
(myeloid) and
mixed colonies at microscope 14 days after the plating. (E) Distribution of
the CD34+ cell
subpopulations and CD34- cells measured by flow cytometry based on the
expression of
hCD133 and hCD90 analysed 4 days after the editing. (F) Representative plots
of the T cell
differentiation stages analysed by flow cytometry 7 weeks after ATO seeding.
(G) HDR
efficiency is measured as proportion of edited alleles in bulk, CD4+ CD8+
double positive (DP)
cells and CD4- CD8- double negative (DN) cells by flow cytometry 6 weeks after
ATO seeding.
DETAILED DESCRIPTION
It must be noted that as used herein and in the appended claims, the singular
forms "a", "an",
and the include plural referents unless the context clearly dictates
otherwise.
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The terms "comprising", "comprises" and "comprised of as used herein are
synonymous with
"including", "includes" or "containing", "contains", and are inclusive or open-
ended and do not
exclude additional, non-recited members, elements or method steps. The terms
"comprising",
"comprises" and "comprised of" also include the term "consisting of".
Numeric ranges are inclusive of the numbers defining the range. Unless
otherwise indicated,
any nucleic acid sequences are written left to right in 5 to 3' orientation;
amino acid sequences
are written left to right in amino to carboxy orientation, respectively.
All recited genomic locations are based on human genome assembly GRCh38.p13
(GCF_000001405.39). One of skill in the art will be able to identify the
corresponding genome
locations in alternative genome assemblies and convert the recited genomic
location
accordingly. For example, RAG1 is located at chr 11: 36510353 to 36579762 in
assembly
GRCh38.p13 and at chr 11: 36532053 to 36601312 in assembly GRCh37.p13.
The publications discussed herein are provided solely for their disclosure
prior to the filing date
of the present application. Nothing herein is to be construed as an admission
that such
publications constitute prior art to the claims appended hereto.
Recombination activating gene 1 (RAG1)
The present invention relates to methods for gene-editing cells to introduce a
RAG1
polypeptide, for example as a treatment for severe combined immunodeficiency.
The present
invention also relates to polynucleotides, vectors, guide RNAs, kits,
compositions, and gene
editing systems for use in said methods, and genomes and cells obtained or
obtainable by
said methods.
"RAG1" is the abbreviated name of the polypeptide encoded by recombination
activating gene
1 and is also known as RAG-1, RN F74, and recombination activating 1.
RAG1 is the catalytic component of the RAG complex, a multiprotein complex
that mediates
the DNA cleavage phase during V(D)J recombination. V(D)J recombination
assembles a
diverse repertoire of immunoglobulin and T-cell receptor genes in developing B
and T-
lymphocytes through rearrangement of different V (variable), in some cases D
(diversity), and
J (joining) gene segments. In the RAG complex, RAG1 mediates the DNA-binding
to the
conserved recombination signal sequences (RSS) and catalyses the DNA cleavage
activities
by introducing a double-strand break between the RSS and the adjacent coding
segment.
RAG2 is not a catalytic component but is required for all known catalytic
activities.
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A "RAG1 polypeptide" is a polypeptide having RAG1 activity, for example a
polypeptide which
is able to form a RAG complex, mediate DNA-binding to the RSS, and introduce a
double-
strand break between the RSS and the adjacent coding segment. Suitably, a RAG1
polypeptide may have the same or similar activity to a wild-type RAG1, e.g.
may have at least
40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at
least 95%, at
least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at
least 150% of
the activity of a wild-type RAG1 polypeptide.
The RAG1 polypeptide may be a fragment of RAG1 and/or a RAG1 variant.
A "fragment of RAG1" may refer to a portion or region of a full-length RAG1
polypeptide that
has the same of similar activity as a full-length RAG1 polypeptide, i.e. the
fragment may be a
functional fragment. The fragment may have at least 40%, at least 50%, at
least 60%, at least
70%, at least 80%, at least 90%, at least 95%, or 100% of the activity of a
full-length RAG1
polypeptide. A person skilled in the art would be able to generate fragments
based on the
known structural and functional features of RAG1. These are described, for
instance, in
Arbuckle, J.L., et al., 2011. BMC biochemistry, 12(1), p.23; Ru, H., et al.,
2015. Cell, 163(5),
pp.1138-1152; and Kim, M.S., et al., 2015. Nature, 518(7540), pp.507-511.
The minimal regions of RAG1 required for catalysis have been identified. These
regions are
referred to as the core proteins. Core RAG1 consists of multiple structural
domains, termed
the nonamer binding domain (NBD; residues 389-464), the central domain
(residues 528-760),
and the C-terminal domain (residues 761-980) domains. Besides the ability to
recognize the
RSS nonamer and heptamer through the NBD and the central domain, respectively,
core
RAG1 contains the essential acidic active site residues (Arbuckle, J.L., et
al., 2011. BMC
biochemistry, 12(1), p.23). Suitably, a fragment of RAG1 comprises the nonamer
binding
domain, the central domain, and/or the C-terminal domain.
A "RAG1 variant" may include an amino acid sequence or a nucleotide sequence
which may
be at least 50%, at least 55%, at least 65%, at least 70%, at least 75%, at
least 80%, at least
85% or at least 90% identical, optionally at least 95% or at least 97% or at
least 99% identical
to a wild-type RAG1 polypeptide. RAG1 variants may have the same or similar
activity to a
wild-type RAG1 polypeptide, e.g. may have at least at least 40%, at least 50%,
at least 60%,
at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at
least 110%, at least
120%, at least 130%, at least 140%, or at least 150% of the activity of a wild-
type RAG1
polypeptide. A person skilled in the art would be able to generate RAG1
variants based on the
known structural and functional features of RAG1 and/or using conservative
substitutions.
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The gene encoding RAG1 (NCB! gene ID: 5896) is located in the human genome at
chr 11:
36510353 to 36579762.
Several alternative mRNAs are transcribed from the RAG1 gene. Transcript
variant 1
(NM_000448) has two exons and one intron. As used herein, the region of the
RAG1 gene
corresponding to the first exon of transcript variant 1 is called the "RAG1
exon 1", the region
of the RAG1 gene corresponding to the intron of transcript variant 1 is called
the "RAG1 intron
1", and the region of the RAG1 gene corresponding to the second exon (which
encodes a
RAG1 polypeptide) is called the "RAG1 exon 2".
Suitably, the RAG1 exon 1 is from chr 11:36568006 to chr 11:36568122; the RAG1
intron 1
is from chr 11: 36568123 to chr 11: 36573290; and/or the RAG1 exon 2 is from
chr 11:
36573291 to chr 11: 36579762.
Suitably, the RAG1 exon 1 consists of the nucleotide sequence of SEQ ID NO: 1,
or variants
thereof; the RAG1 intron 1 consists of the nucleotide sequence of SEQ ID NO:
2, or variants
thereof; and/or the RAG1 exon 2 consists of the nucleotide sequence of SEQ ID
NO: 3, or
variants thereof.
Illustrative RAG1 exon 1 (SEQ ID NO: 1)
agaaacaagagggcaaggagagagcagagaacacactttgccttctctttggtattgagtaatatcaaccaaattgc
agacatctcaacactttggccaggcagcctgctgagcaag
Illustrative RAG1 intron 1 (SEQ ID NO: 2)
gtaacactcatacifitcatgccttgagccaaaatatttattacatttttatglitctaactagaagtgcttgagctti
fittccttcc
aggtgatgaggggatggaatgagcaaagctacatcaatttttttttaatgtatgaaaataaaaaaggtacaagaggcc
aagtttagggccactgaaggttcatagaaagatgcaaaatatctgaattactataaatgaatgctattgtcagaggaaa
ggtttaaggagtgcttcttgaatgaatgtgtacaaatcagcagaaggtaaggtgtgagactcttggaaatgaatactgg
t
agttcaggtgagaaaaataatcaggaacataatagggtgggaggaaatgtatggtttcccaggtattaacaagtattg
ccaggcatttcctgaactagattggcctaagtaggagaccaatgtttctcaaaatattcactcattttagaatcactga
atg
tttaaaaatgcaatttctggattccttcccaaacagccagactctligggacctgatgatctgcatttctlittaaaaa
caaa
ctcgctcatgattctgatttgtattaattttgagaattgccatggtagagaccctgctttgaggttatgttcttgagtc
aggattc
ctggccagggattgtgatgatatatttctetttctgaagtggttcatgcaagaggttgtctgaaggaagagcaagaatt
gt
agtgttattttgtggatacttgagacttataaaaaggctttttattttgtcacatttttgatacatgatgtttggcaaa
aaacaga
cgatagtatttgcagagtgaatgaataagtggaacaggtgtgataatgagaggtcacacttgagcacacagttattact
tggaaattgtgtacagactaagttgaagatgttaggagggaagattgtgggccaagtaacggggtgtatgtgtgtgggt
atagggtgggcagctgggatggaaatggggggctgctgctgctgctgcaccctggcctcctgaactaatgatatcact
caccagaaactactgttcctgcactgtccaagccaccccaaactagtttgtcaaaatgaatctgtgctgtgtggaggga
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ggcacgcctgtagctctgatgtcagatggcaatgtcgagatggcagtggccggtggggacagggctgagccagcac
caaccactcagcctttgagatcccgaggctggtctactgctgagaccttttgttagaagagaggagatcaagcatttgc
aaggtttctgagtgtcaaaatatgaatccaagataactctttcacaatcctaacttcatgctgtctacaggtccatatt
ttag
cctgctttctccatgttcatccgaaaagaaagaaaagctaagggtggtggtcatatttgaaattagccagatcttaagt
ttt
tctgggggaaatttagaagaaaatatggaaaagtgactatgagcacatatacagctagtctttaaaacagttttatcca
aaataaatgtatcacaaaattaataaaaatagttacttg
cttgttttgaataattcaaatgatacaaaaattaataaaataa
aaagtgcaaaaggccctcttatcaatgccaattctattffittcagaaattaaacactgttaagattttagtgtgtatc
ctttca
gaattcctgtgatttcatatatgtacaaatacaaacgtatctacataaagggaatcctactatacttgctattgtcatt
ctattc
tctgctlittcatgtgagcatctttccatgtcactgatgcatacagaaattg
cacatatgcatcagtgcatacagaaaattaa
attttctgcatggttttccactgtatgtctggaccatagtttatttaataataatgccctttgggtaattatttatatt
gtttcctg cttt
ttcaaagtaacagcttttgaaacaaatctctctctgtctttatataaatattgttgcattcctgtggaaatgtttctat
tggataa
cttcccaaaaggagatttattgcatcaaagataatatattcaaaaattttaaagatattgctaaattgtctagtaggta
tttta
taccaatttatactcctcccaagaatgtatggagatatcttaatttctccatgccttcattaatgctgaaccatataag
tagttt
taatctttgctaattgaatagataaaaaatatctaatctaagtctagttcttaaaagttctatcttctaccaaaagtaa
tacac
gtctattttagggagtaaaaatcacaagtaaggataaaaaatagtgcagcaataaacacaggagtgtagatgtctctg
aacatactg atttaacttcctttgg ataaatacccagtagtagg actgctgg
atcatataataattctatctttag tffitttgag
gacctccatactattcttcatagtggctgtactaatttacattcctaccaactgtgtatgaaggttcccttttctctac
atccttg
ccagcattcattattgcttgtcatttggatacaatctattttaactggggtgagatgacatctcattgtagttttgata
tgcatttc
tctgatgatcagtggtgttgagcaccttlicatatacctgtttgccatttgtatgtcttcctttgagaaatgtctattc
agatatttt
acctattttaaaatcggattattagattgtttcctgtagagttgtttgagctccttgtatattctggttattaatctct
tgtcagatgc
atagcttacaaatattttctcccatcatgtg gattgtgtcttca ctttgtgg attgtttactttgctgtg cag
aagcttttaacttg a
tgcaatcccatttgtccacttttgctttggttgccttccacaggagtatttaaataaatgtagtttggtagattttggt
atagtaat
gcaggccagtgggagtcaggggagaaatgtgtagggaagtgagatagttctaaggatcctacaaacatgccttatga
ttgacttactcaatgtgaaagtcaatattaaacttgatgagctctagagatggtcatgcattttaaaaagaattactca
aaa
tattgtcttggaataccagagagcaagtgctttaagtataggctgggaagtaaaatgctaaaggaatgagaaggcattt
ggggttgagttcaacctaagagg caggggagccacagggaaagacctagcacctg ccacagaagagaattagg
aagcagaattgaactataagcaattttgaggtgttcgttgggctgcagttgaaatatttlitgaggttaatgagacatt
tgaa
atg gccgtgtattgtttaactcttg catagtcctgcatag gg aacaatctaatag gatttctctgtg
aatcaagtcttag aa a
tttgcttttaatttttatgaaaaacgcccatttctttgtttttg
agacagagtcctgctctgtcatccaggctgggttgcagtggc
gtgatcttggcccactgcaatctctgcctcctgggttcaggcaattttcctgtctcagcctcccgagtagctgggattt
caa
gtgcctgccaccatgcccggctaaatttttttgtatttttggtacagatggagtatcaccatgttggccaggctggtct
cgaa
ctcctgacctcaagtgattcaccagccttgacctcccaaagtgttgggatcacaggcatgagccactgtgcctgtgccc
caaaacaccaatttctgatgtgtgatgcatgtaagatagaacaaacttcagtaaagcggggacttgaaaagaggcttt
ggtaacagctgtcagcattaacccttgcccctccgtacctcctaatcccacccctgctcaaagtatgttcatctgagaa
ttt
gtctccataactatgtgactataaaaattctcatcgattttgttagttgatcaattgagggaaaaacatatgttacttg
atata
actggtgggtcaaaagaattaacccaggcaaatttgagataggtggatggg
atgatggattgaaaatacagctgctct
ctttccaatcatgtactaagtaatttgggaaagattgatctaattgggtctagagagtacacttcacatggcattgttt
gactt
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tttttctgcatcgctagcgatctgtgcattacaactcaaatcagtcgggtttcctggcatatgtaattgccaatgtttt
ttacca
gaag ag aaacattactcccacctcttcttattatgttacaaactatagtg
ctaatgaccatcgaccaacagtgactttcag
g atg acctgtgtg agttttatctg aaaccatgtgaatttttcatcttaaaagtcccttag
aatctcagtctatgtacactcaggt
ttgttgcaggtttagagttccgtgffitttgtttctaatgtagacacagecttataatttacaacagcattcactaatt
aaaattgt
aagcataattactatccacgatacttattattagtttgcattcataaagctcaaaattcacttcatcctttcaagtagt
gaata
attagtttctttgggtttgcagctttatcatccttttatgacccatttggaagaaataaacaaccaaccccctggaaga
ctgc
tttaaaaagctggaaatacattgtccagctagtacaatgaggctaatacaatgtggaaaatattacttttctttgattt
tagt
agcctglltatctttacatttactgaacaaataactattgagcacctaatgtatactgggacccttggggaggcaaaga
tg
aatcaaag attctgtccttaaagaccttaag gtttttgtgg aagg aaataaaactttacatgtatatatttaag
cacttatat
gtgtgtaacaggtataagtaaccataaacactgtcagaag aggaaataactctatgatcag
cacctaacatgatatatt
aaggtag aagatttaatacatatcttttgg aatacatg aataaataattg
aatgtatttatttttattatttataagatacatca
gtgggatattgatattggtcttaatatgacttgtfficattglictcag
Illustrative RAG1 exon 2 (SEQ ID NO: 3)
gtacctcagccagcATGGCAGCCTCTTTCCCACCCACCTTGGGACTCAGTTCTGCCCC
AGATGAAATTCAGCACCCACATATTAAATTTTCAGAATGGAAATTTAAGCTGTTC
CGGGTGAGATCCTTTGAAAAGACACCTGAAGAAGCTCAAAAGGAAAAGAAGGAT
TCCTTTGAGGGGAAACCCTCTCTGGAGCAATCTCCAGCAGTCCTGGACAAGGC
TGATGGTCAGAAGCCAGTC CCAACTCAGC CATTGTTAAAAGCCCACCCTAAGTT
TTCAAAGAAATTTCACGACAACGAGAAAGCAAGAGGCAAAGCGATCCATCAAGC
CAACCTTC GACATCTCTGCCGCATCTGTGGGAATTCTTTTA GAG CTGATGAGCA
CAACAGGAGATATCCAGTCCATGGTCCTGTGGATGGTAAAACCCTAGGCCTTTT
ACGAAAGAAGGAAAAGAGAGCTACTTCCTGGCCGGACCTCATTGCCAAGGTTTT
CCGGATCGATGTGAAGGCAGATGTTGACTCGATCCACCCCACTGAGTTCTGCC
ATAACTGCTGGAGCATCATGCACAGGAAGTTTAGCAGTGCCCCATGTGAGGTTT
ACTTCCCGAGGAACGTGACCATGGAGTGGCACCCCCACACACCATCCTGTGAC
ATCTGCAACACTGCCCGTCGGGGACTCAAGAGGAAGAGTOTTCAGCCAAACTT
GCAGCTCAGCAAAAAACTCAAAACTGTGCTTGACCAAGCAAGACAAGCCCGTCA
GCACAAGAGAAGAGCTCAGGCAAGGATCAGCAGCAAGGATGTCATGAAGAAGA
TCGCCAACTGCAGTAAGATACATCTTAGTACCAAGCTCCTTGCAGTGGACTTCC
CAGAGCACTTTGTGAAATCCATCTCCTGCCAGATCTGTGAACACATTCTGGCTG
ACCCTGTGGAGACCAACTGTAAGCATGTCTTTTGCCGGGTCTGCATTCTCAGAT
GCCTCAAAGTCATGGGCAGCTATTGTCCCTCTTGCCGATATCCATGCTTCCCTA
CTGACCTGGAGAGTCCAGTGAAGTCCTTTCTGAGCGTCTTGAATTCCCTGATGG
TGAAATGTCCAGCAAAAGAGTGCAATGAGGAGGTCAGTTTGGAAAAATATAATC
ACCACATCTCAAGTCACAAGGAATCAAAAGAGATTTTTGTGCACATTAATAAAGG
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GGGCCGGCCCCGCCAACATCTTCTGTCGCTGACTCGGAGAGCTCAGAAGCACC
GGCTGAGGGAGCTCAAGCTGCAAGTCAAAGCCTTTGCTGACAAAGAAGAAGGT
GGAGATGTGAAGTCCGTGTGCATGACCTTGTTCCTGCTGGCTCTGAGGGCGAG
GAATGAGCACAGGCAAGCTGATGAGCTGGAGGCCATCATGCAGGGAAAGGGCT
CTGGCCTGCAGCCAGCTGTTTGCTTGGCCATCCGTGTCAACACCTTCCTCAGCT
GCAGTCAGTACCACAAGATGTACAGGACTGTGAAAGCCATCACAGGGAGACAG
ATTTTTCAGCCTTTGCATGCCCTTCGGAATGCTGAGAAGGTACTTCTGCCAGGC
TACCACCACTTTGAGTGGCAGCCACCTCTGAAGAATGTGTCTTCCAGCACTGAT
GTTGGCATTATTGATGGGCTGTCTGGACTATCATCCTCTGTGGATGATTACCCA
GTGGACACCATTGCAAAGAGGTTCCGCTATGATTCAGCTTTGGTGTCTGCTTTG
ATGGACATGGAAGAAGACATCTTGGAAGGCATGAGATCCCAAGACCTTGATGAT
TACCTGAATGGCCCCTTCACTGTGGTGGTGAAGGAGTCTTGTGATGGAATGGG
AGACGTGAGTGAGAAGCATGGGAGTGGGCCTGTAGTTCCAGAAAAGGCAGTCC
GTTTTTCATTCACAATCATGAAAATTACTATTGCCCACAGCTCTCAGAATGTGAA
AGTATTTGAAGAAGCCAAACCTAACTCTGAACTGTGTTGCAAGCCATTGTGCCTT
ATGCTGGCAGATGAGTCTGACCACGAGACGCTGACTGCCATCCTGAGTCCTCT
CATTGCTGAGAGGGAGGCCATGAAGAGCAGTGAATTAATGCTTGAGCTGGGAG
GCATTCTCCGGACTTTCAAGTTCATCTTCAGGGGCACCGGCTATGATGAAAAAC
TTGTGCGGGAAGTGGAAGGCCTCGAGGCTTCTGG CTCAGTCTACATTTGTACTC
TTTGTGATGCCACCCGTCTGGAAG CCTCTCAAAATCTTGTCTTCCACTCTATAAC
CAGAAGCCATGCTGAGAACCTGGAACGTTATGAGGTCTGGCGTTCCAACCCTTA
CCATGAGTCTGTGGAAGAACTGCGGGATCGGGTGAAAGGGGTCTCAGCTAAAC
CTTTCATTGAGACAGTCCCITCCATAGATGCACTCCACTGTGACATTGGCAATG
CAGCTGAGTTCTACAAGATCTTCCAGCTAGAGATAGGGGAAGTGTATAAGAATC
CCAATGCTTCCAAAGAGGAAAGGAAAAGGTGGCAGGCCACACTGGACAAGCAT
CTCCGGAAGAAGATGAACCTCAAACCAATCATGAGGATGAATGGCAACTITGCC
AGGAAGCTCATGACCAAAGAGACTGTGGATGCAGTTTGTGAGTTAATTCCTTCC
GAGGAGAGGCACGAGGCTCTGAGGGAGCTGATGGATCTTTACCTGAAGATGAA
ACCAGTATGGCGATCATCATGCCCTGCTAAAGAGTGCCCAGAATCCCTCTGCCA
GTACAGTTICAATTCACAGCGTTTTGCTGAGCTCCTTTCTACGAAGTTCAAGTAT
AGGTATGAGGGAAAAATCACCAATTATTTICACAAAACCCTGGCCCATGTTCCTG
AAATTATTGAGAGGGATGGCTCCATTGGGGCATGGGCAAGTGAGGGAAATGAG
TCTGGTAACAAACTGTTTAGGCGCTTCCGGAAAATGAATGCCAGGCAGTCCAAA
TGCTATGAGATGGAAGATGTCCTGAAACACCACTGGTTGTACACCTCCAAATAC
CTCCAGAAGTTTATGAATGCTCATAATGCATTAAAAACCTCTGGGTTTACCATGA
ACCCTCAGGCAAGCTTAGGGGACCCATTAGGCATAGAGGACTCTCTGGAAAGC
CAAGATTCAATGGAATTTTAAgtagg gcaaccacttatg agttg gtttttgcaattg agtttccctctgggttg
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cattgagggcttctcctagcaccctttactgctgtgtatggggcttcaccatccaagaggtggtaggttggagtaagat
gc
tacagatg ctctcaagtcaggaatagaaactgatgagctgattg
cttgaggcttttagtgagttccgaaaagcaacagg
aaaaatcagttatctgaaag ctcagtaactcagaacaggagtaactg caggggaccagagatgagcaaagatctgt
gtgtgttggggagctgtcatgtaaatcaaagccaaggttgtcaaagaacagccagtgaggccaggaaagaaattggt
cttgtg gttttcatttttttcccccttg
attgattatattttgtattgagatatgataagtgccttctatttcatttttgaataattcttcatt
tttataattttacatatcttggcttgctatataagattcaaaagagctttttaaattffictaataatatcttacattt
gtacagcatg
atgacctttacaaagtgctctcaatgcatttacccattcgttatataaatatgttacatcaggacaactttgagaaaat
cagt
cctlitttatgtttaaattatgtatctattgtaaccttcagagtttaggaggtcatctgctgtcatggatttttcaata
atgaatttag
aatacacctgttagctacagttagttattaaatcttctgataatatatgtttacttagctatcagaagccaagtatgat
tclitat
ttttactttttcatttcaagaaatttagagtttccaaatttagagclictgcatacagtcttaaagccacagaggcttg
taaaa
atataggttagcttgatgtctaaaaatatatttcatgtcttactgaaacattttgccagactttctccaaatgaaacct
gaatc
aattffictaaatctaggtttcatag
agtcctctcctctgcaatgtgttattctlictataatgatcagtttactttcagtgg attcag
aattgtgtagcaggataaccttgtatttttccatccgctaagtttagatggagtccaaacgcagtacagcagaagagtt
a
acatttacacagtgctttttaccactgtgg aatgttttcacactcatttttccttacaacaattctgagg
agtaggtgttgttatta
tctccatttgatgggggtttaaatgatttgctcaaagtcatttaggggtaataaatacttggcttggaaatttaacaca
gtcct
tttgtctccaaagcccttcttctttccaccacaaattaatcactatgtttataaggtagtatcagaatttttttaggat
tcacaac
taatcactatag cacatgaccttgggattacatttttatgg
ggcaggggtaagcaagtttttaaatcatttgtgtgctctggct
cttttgatagaagaaagcaacacaaaagctccaaagggccccctaaccctcttgtggctccagttatttggaaactatg
atctgcatccttaggaatctgggatttgccagttgctggcaatgtagagcaggcatggaattttatatgctagtgagtc
ata
atg atatgttagtgttaattagttttttcttcctttg attttattg gccataattg
ctactcttcatacacagtatatcaaag ag cttg
ataatttagttgtcaaaagtgcatcggcgacattatctttaattgtatgtatttggtgcttcttcagggattgaactca
gtatcttt
cattaaaaaacacagcagttttccttg ctttttatatgcagaatatcaaagtcatttctaatttagttgtcaaaa
acatataca
tattttaacattagttffittgaaaactcttgglittglitlittggaaatgagtgggccactaagccacactttccct
tcatcctgct
taatccttccagcatgtctctgcactaataaacagctaaattcacataatcatcctatttactgaagcatggtcatgct
ggtt
tatagatttlitacccatttctactclitttctctattggtggcactgtaaatactttccagtattaaattatcctifi
ctaacactgta
ggaactattttgaatgcatgtgactaagagcatgatttatagcacaacctttccaataatcccttaatcagatcacatt
ttga
taaaccctgggaacatctggctgcaggaatttcaatatgtagaaacgctgcctatggttttttgcccttactgttgaga
ctg
caatatcctagaccctagttttatactagagttttatttttagcaatgcctattgcaagtgcaattatatactccaggg
aaattc
accacactgaatcg agcatttgtgtgtgtatgtgtg aa gtatatactg gg acttcag
aagtgcaatgtatlittctcctgtg a
aacctgaatctacaagttttcctgccaagccactcaggtgcattgcagggaccagtgataatggctgatgaaaattgat
gattggtcagtgaggtcaaaaggagccttgggattaataaacatg cactgagaag caagaggaggagaaaaagat
gtcffittcttccaggtgaactggaatttagttttgcctcagatttlittcccacaagatacagaagaagataaag
atttlittgg
ttgagagtgtgggtcttgcattacatcaaacagagttcaaattccacacagataagaggcagg
atatataagcgccag
tggtagttgggaggaataaaccattatttggatgcaggtggtttttgattgcaaatatgtgtgtgtcttcagtgattgt
atgac
agatgatgtattcttttgatgttaaaagattttaagtaag
agtagatacattgtacccattttacattttcttattttaactacagt
aatctacataaatatacctcag aaatcatttttg gtgattattttttgttttgtag aattg cacttcag
tttattttcttacaaataac
cttacattllgtttaatggcttccaagagcctttttffittttgtatttcagagaaaattcaggtaccaggatgcaatg
gatttattt
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gattcaggggacctgtgtttccatgtcaaatgtlitcaaataaaatgaaatatgagificaatactifitatattttaa
tatttcca
ttcattaatattatggttattgtcagcaattttatgtttgaatatttgaaataaaagtttaagatttgaaaa
In the illustrative RAG1 exon 2 (SEQ ID NO: 3), upper case letters indicate a
nucleotide
sequence which encodes a RAG1 polypeptide.
RAG1 polypeptides
The RAG1 polypeptide may be a human RAG1 polypeptide. Suitably, the RAG1
polypeptide
may comprise or consist of a polypeptide sequence of UniProtKB accession
P15918, or a
fragment or variant thereof.
In some embodiments of the invention, the RAG1 polypeptide comprises or
consists of an
amino acid sequence which is at least 70% identical to SEQ ID NO: 4 or a
fragment thereof.
Suitably, the RAG1 polypeptide comprises or consists of an amino acid sequence
which is at
least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least
99% identical to
SEQ ID NO: 4 or a fragment thereof.
In some embodiments, the RAG1 polypeptide comprises or consists of SEQ ID NO:
4 or a
fragment thereof.
RAG1 polypeptide isoform 1, UniProtKB accession P15918 (SEQ ID NO: 4)
MAASFPPTLGLSSAPDEIQHPHIKFSEWKFKLFRVRSFEKTPEEAQKEKKDSFEGKP
SLEQSPAVLDKADGQKPVPTQPLLKAHPKFSKKFHDNEKARGKAIHQANLRHLCRI
CGNSFRADEHNRRYPVHGPVDGKTLGLLRKKEKRATSWPDLIAKVFRIDVKADVDS
I HPTEFCHNCWSIMHRKFSSAPCEVYFPRNVTM EWHPHTPSCDICNTARRGLKRKS
LQPNLQLSKKLKTVLDQARQARQHKRRAQARISSKDVMKKIANCSKI HLSTKLLAVD
FPEHFVKSISCQICEHILADPVETNCKHVFCRVCILRCLKVMGSYCPSCRYPCFPTDL
ESPVKSFLSVLNSLMVKCPAKECNEEVSLEKYN HHISSHKESKEI FVHINKGGRPRQ
HLLSLTRRAQKHRLRELKLQVKAFADKEEGGDVKSVCMTLFLLALRARNEHRQADE
LEAIMQGKGSGLQPAVCLAIRVNTFLSCSQYHKMYRTVKAITGRQIFQPLHALRNAE
KVLLPGYHHFEWQPPLKNVSSSTDVGIIDGLSGLSSSVDDYPVDTIAKRFRYDSALV
SALMDMEEDI LEGMRSQDLDDYLNGPFTVVVKESCDGMGDVSEKHGSGPVVPEK
AVRFSFTIMKITIAHSSQNVKVFEEAKPNSELCCKPLCLMLADESDHETLTAILSPLIA
EREAMKSSELMLELGGILRTFKFIFRGTGYDEKLVR EVEGLEASGSVYICTLCDATRL
EASQNLVFHSITRSHAENLERYEVWRSNPYHESVEELRDRVKGVSAKPFIETVPSID
ALHCDIGNAAEFYKIFQLEIGEVYKNPNASKEERKRWQATLDKHLRKKM NLKPIMRM
NGNFARKLMTKETVDAVCELI PSEERHEALRELMDLYLKMKPVWRSSCPAKECPES
LCQYSFNSQRFAELLSTKFKYRYEGKITNYFHKTLAHVPEI IERDGSIGAWASEGNE
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SGNKLFRRFRKMNARQSKCYEMEDVLKHHWLYTSKYLQKFMNAHNALKTSGFTM
NPQASLGDPLGIEDSLESQDSMEF
In some embodiments of the invention, the RAG1 polypeptide comprises or
consists of an
amino acid sequence which is at least 70% identical to SEQ ID NO: 5 or a
fragment thereof.
Suitably, the RAG1 polypeptide comprises or consists of an amino acid sequence
which is at
least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least
99% identical to
SEQ ID NO: 5 or a fragment thereof.
In some embodiments, the RAG1 polypeptide comprises or consists of SEQ ID NO:
5 or a
fragment thereof.
RAG1 polypeptide iso form 2, UniProtKB accession P15918 (SEQ ID NO: 5)
MAASFPPTLGLSSAPDEIQHPHIKFSEWKFKLFRVRSFEKTPEEAQKEKKDSFEGKP
SLEQSPAVLDKADGQKPVPTOPLLKAHPKFSKKFHDNEKARGKAIHQANLRHLCRI
CGNSFRADEHNRRYPVHGPVDGKTLGLLRKKEKRATSWPDLIAKVFRIDVKADVDS
IHPTEFCHNCWSIMHRKFSSAPCEVYFPRNVTMEWHPHTPSCDICNTARRGLKRKS
LQPNLQLSKKLKTVLDQARQARQHKRRAQARISSKDVMKKIANCSKIHLSTKLLAVD
FPEHFVKSISCQICEHILADPVETNCKHVFCRVCILRCLKVMGSYCPSCRYPCFPTDL
ESPVKSFLSVLNSLMVKCPAKECNEEVSLEKYNHHISSHKESKEIFVHINKGGRPRQ
HLLSLTRRAQKHRLRELKLQVKAFADKEEGGDVKSVCMTLFLLALRARNEHRQADE
LEAIMQGKGSGLQPAVCLAIRVNTFLSCSQYHKMYRTVKAITGRQIFQPLHALRNAE
KVLLPGYHHFEWQPPLKNVSSSTDVGIIDGLSGLSSSVDDYPVDTIAKRFRYDSALV
SALMDMEEDILEGMRSQDLDDYLNGPFTVVVKESCDGMGDVSEKHGSGPVVPEK
AVRFSFTIMKITIAHSSQNVKVFEEAKPNSELCCKPLCLMLADESDHETLTAILSPLIA
EREAMKSSELMLELGGILRTFKFIFRGTGYDEKLVREVEGLEASGSVYICTLCDATRL
EASQNLVFHSITRSHAENLERYEVWRSNPYHESVEELRDRVKGVSAKPFIETVPSID
ALHCDIGNAAEFYKIFQLEIGEVYKNPNASKEERKRWQATLDKHLRKKMNLKPIMRM
NGNFARKLMTKETVDAVCELIPSEERHEALRELMDLYLKMKPVWRSSCPAKECPES
LCQYSFNSQRFAELLSTKFKYRN
RAG1 polvnucleotides
The nucleotide sequence encoding a RAG1 polypeptide may be codon-optimised.
Suitably,
the nucleotide sequence encoding a RAG1 polypeptide may be codon optimised for
expression in a human cell.
Different cells differ in their usage of particular codons. This codon bias
corresponds to a bias
in the relative abundance of particular tRNAs in the cell type. By altering
the codons in the
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sequence so that they are tailored to match with the relative abundance of
corresponding
tRNAs, it is possible to increase expression. By the same token, it is
possible to decrease
expression by deliberately choosing codons for which the corresponding tRNAs
are known to
be rare in the particular cell type. Thus, an additional degree of
translational control is
available. Codon usage tables are known in the art for mammalian cells (e.g.
humans), as well
as for a variety of other organisms.
In some embodiments of the invention, the nucleotide sequence encoding a RAG1
polypeptide
comprises or consists of a nucleotide sequence which is at least 70% identical
to SEQ ID NO:
6 or a fragment thereof. Suitably, the nucleotide sequence encoding a RAG1
polypeptide
comprises or consists of a nucleotide sequence which is at least 80%, at least
85%, at least
90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 6 or a
fragment
thereof.
In some embodiments of the invention, the nucleotide sequence encoding a RAG1
polypeptide
comprises or consists of the nucleotide sequence SEQ ID NO: 6 or a fragment
thereof.
Exemplary nucleotide sequence encoding a RAGI polypeptide (SEQ ID NO: 6)
atggccgcctccttcccacctacccttggattgtcctccgcccctgacgaaattcaacatccccacatcaaattctcgg
a
gtggaaglicaagctclitcgcgtgcgctcgttcgaaaagacccccgaggaagcccaaaaggagaagaaagactc
attcgaaggaaaacccagcctcgaacagtccccggccgtcctggacaaggccgacgggcagaagcctgtgccga
cccagccgctgctgaaagcgcacccgaaattctccaagaagtttcacgataacgagaaggcccggggaaaggcc
atccaccaagcaaaccttagacacctgtgccgcatctgtgggaactcattcagagccgacgaacataaccggagat
accctgtgcatggccctgtcgacggaaagaccctggggctcctgagaaagaaggagaagagggcgacatcctgg
ccggacctgatcgcaaaggtgttcagaatcgacgtgaaggcagatgtggacagcatccacccaaccgagttctgcc
acaactgctggagcattatgcaccggaagttcagctcagcgccctgtgaagtgtacttcccgcgcaacgtgactatgg
agtggcatccacacactccgtcctgcgacatctgtaacactgctcggcgcggactcaagaggaagtccctgcagccg
aatctgcagctgagcaagaagcttaagaccgtgctggaccaggctcggcaggcccgccagcacaagcgacgcgc
ccaggcccggatctcatctaaggatgtgatgaagaagatcgccaattgcagcaaaatccacctgtctaccaagctgct
ggcggtggacttcccggagcacttcgtgaagtccatcagctgtcagatctgcgagcatattctcgccgaccccgtgga
gactaattgcaagcacgtgttctgccgcgtgtgcatcctgcgctgcctgaaggtcatgggctcctattgcccttcctgc
cg
gtacccctgtttccctactgatctggagtccccggtcaagtccttcttgtccgtgctgaactccctgatggtcaaatgt
cccg
caaaggagtgcaatgaggaagtgtccctggaaaagtacaaccaccacatcagcagccacaaggagtccaaaga
aatctttgtgcacattaacaagggcggtcggccccggcagcatctgctctcgctgactcgccgggcccagaagcaca
ggctccgggagctgaagctgcaagtcaaggccttcgccgacaaggaagagggaggagatgtgaagtccgtgtgca
tgaccctgtttttgctggcgctgcgggctcggaacgaacacagacaagctgatgaactggaggccatcatgcagggc
aaaggatcgggactccagccggctgtgtgtctcgccatccgcgtcaacacattcctctcatgctcccaataccacaag
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atgtacaggactgtgaaggccatcaccggacggcagatclitcagccactccacgcccttcggaacgcagaaaagg
tcttgctgccgggataccatcatttcgaatggcagccgcccttgaaaaacgtgtcctcgtccaccgacgtgggcattat
t
gatgggctgagcggcctgtcctcctctgtggatgactaccctgtggataccatcgccaaacggttcagatacgattccg
cgctggtgtcggccctgatggacatggaggaggacatcctggagggaatgagatcacaagatctggacgactacct
caacgggcccttcacggtggtggtcaaggaatcgtgcgatggaatgggcgacgtgtcggagaagcacggttccgga
cctgtggtgccggaaaaggccgtgcgcttctccttcaccatcatgaagatcaccattgcgcatagctcccagaacgtca
aagtgttcgaagaggccaagccgaactcagagctctgctgcaagccgctgtgcctgatgttggcggacgagagcga
tcacgaaaccctgaccgccattctgtcgcctctgatcgcggagagggaggccatgaagtcctccgaactgatgctgg
agctgggcggtattttgcggactlitaagttcatcttccggggaaccggttatgacgaaaagctcgtgcgcgaagtgga
gggcctggaagcctcaggctccgtctacatctgcactctctgcgacgccacccggctggaggcgtcacagaatcttgt
gttccactcgatcactaggtcccacgcggagaacctggaacgctatgaggtctggcgctctaacccataccacgaatc
cgtggaagaacttcgggacagagtgaagggagtgtcagcaaagccificattgaaaccgtgcctagcatcgacgcc
ctccattgcgacatcggcaacgccgccgagttctacaagatcttccagcttgagatcggggaagtgtacaagaaccc
gaacgcctccaaggaagaaagaaagcggtggcaggctacccttgacaaacacctccgcaagaagatgaacctg
aagcccattatgcggatgaacggaaacttcgctaggaagctgatgactaaggaaacggtcgacgcggtctgtgaact
gatccccagcgaagaacgacatgaagcgctgcgcgaactcatggacctgtacctgaagatgaagcctgtctggcgg
agctcgtgccctgccaaggagtgcccggagtcgctgtgtcagtacagctttaacagccaaaggttcgcagagctgctg
tcgaccaagttcaagtacagatacgaaggaaagattaccaactacttccacaagactctcgctcacgtgcccgagatt
atcgaacgcgatggttccatcggggcctgggcctccgagggcaacgagtcgggcaacaagttgliccgccgglitag
aaagatgaacgcccgccagtccaagtgctacgaaatggaagatgtgctgaagcatcactggctgtatacctccaagt
acctccagaagttcatgaacgcacataacgccctcaagacctccgggttcaccatgaacccccaggcctccctcggt
gaccctctgggaattgaagatagcttggagagccaggactcgatggaattcta
Polynucleotides and genomes
In one aspect, the present invention provides a polynucleotide comprising from
5' to 3': a first
homology region, a splice acceptor sequence, a nucleotide sequence encoding a
RAG1
polypeptide, and a second homology region. The polynucleotide may be an
isolated
polynucleotide. The polynucleotide may be a DNA molecule, e.g. a double-
stranded DNA
molecule.
Suitably, the polynucleotide of the invention may be limited to a size
suitable to be inserted
into a vector (e.g. an adeno-associated viral (AAV) vector, such as AAV6).
Suitably, the
polynucleotide of the invention may be 5.0 kb or less, 4.9 kb or less, 4.8 kb
or less, 4.7 kb or
less, 4.6 kb or less, 4.5 kb or less, 4.4 kb or less, 4.3 kb or less, 4.2 kb
or less, 4.1 kb or less,
4.0 kb or less in total size. In some embodiments, the polynucleotide of the
invention is 4.1 kb
or less or 4.0 kb or less in size.
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In another aspect, the present invention provides a genome comprising a splice
acceptor
sequence and a nucleotide sequence encoding a RAG1 polypeptide. Suitably, the
genome
may comprise the polynucleotide of the present invention. The genome may be an
isolated
genome. The genome may be a mammalian genome, e.g. a human genome.
Homology regions
A "homology region" (also known as "homology arm") is a nucleotide sequence
which is
located upstream or downstream of a nucleotide sequence to be inserted (a
"nucleotide
sequence insert" e.g a splice acceptor sequence and a nucleotide sequence
encoding a
RAG1 polypeptide). The polynucleotide of the present invention comprises two
homology
regions, one upstream of the nucleotide sequence insert (the "first homology
region") and one
downstream of the nucleotide insert (the "second homology region").
Each "homology region" is designed such that the nucleotide sequence insert
can be
introduced into a genome at a site of a double strand break (DSB) by homology-
directed repair
(HDR). One of skill in the art will be able to design homology arms depending
on the desired
insertion site (i.e. the site of the DSB) (see e.g. Ran, F.A., et al., 2013.
Nature protocols, 8(11),
pp.2281-2308). Each "homology region" is homologous to a region either side of
the DSB. For
example, the first homology region may be homologous to a region upstream of
the DSB and
the second homology region may be homologous to a region downstream of the
DSB.
As used herein, the term "homologous" means that the nucleotide sequences are
similar or
identical. For example, the nucleotide sequences may be at least 70%
identical, at least 75%
identical, at least 80% identical, at least 85% identical, at least 90%
identical, at least 95%
identical, at least 98% identical, at least 99% identical, or 100% identical.
As used herein, "upstream" and "downstream" both refer to relative positions
in DNA or RNA.
Each strand of DNA or RNA has a 5' end and a 3' end and, by convention,
"upstream" and
"downstream" relate to the 5 to 3' direction respectively in which RNA
transcription takes
place. For example, when considering double-stranded DNA, "upstream" is toward
the 5' end
of the coding strand for the gene in question (e.g. RAG1) and downstream is
toward the 3'
end of the coding strand for the gene in question (e.g. RAG1).
The homology regions may be any length suitable for HDR. The homology regions
may be
the same or different lengths. Suitably, the homology regions are each
independently 50-1000
bp in length, 100-500 bp in length, or 200-400 bp in length. For example, the
first homology
may be 50-1000 bp in length and homologous to a region upstream of a DSB and
the second
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homology region may be 50-1000 bp in length and homologous to a region
downstream of the
DSB.
In some embodiments:
(i) the first homology region is homologous to a first region of the RAG1
intron 1 and
the second homology region is homologous to a second region of the RAG1 intron
1;
or
(ii) the first homology region is homologous to a first region of the RAG1
intron 1 or the
RAG1 exon 2 and the second homology region is homologous to a second region of
the RAG1 exon 2.
In some embodiments, the first homology region is homologous to a first region
of the RAG1
intron 1 and the second homology region is homologous to a second region of
the RAG1 intron
1.
In some embodiments:
(i) the first homology region is homologous to a region upstream of chr 11:
36569295
and the second homology region is homologous to a region downstream of chr 11:
36569298;
(ii) the first homology region is homologous to a region upstream of chr 11:
36573790
and the second homology region is homologous to a region downstream of chr 11:
36573793;
(iii) the first homology region is homologous to a region upstream of chr 11:
36573641
and the second homology region is homologous to a region downstream of chr 11:
36573644;
(iv) the first homology region is homologous to a region upstream of chr 11:
36573351
and the second homology region is homologous to a region downstream of chr 11:
36573354;
(v) the first homology region is homologous to a region upstream of chr 11:
36569080
and the second homology region is homologous to a region downstream of chr 11:
36569083;
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(vi) the first homology region is homologous to a region upstream of chr 11:
36572472
and the second homology region is homologous to a region downstream of chr 11:
36572475;
(vii) the first homology region is homologous to a region upstream of chr 11:
36571458
and the second homology region is homologous to a region downstream of chr 11:
36571461;
(viii) the first homology region is homologous to a region upstream of chr 11:
36571366
and the second homology region is homologous to a region downstream of chr 11:
36571369;
(ix) the first homology region is homologous to a region upstream of chr 11:
36572859
and the second homology region is homologous to a region downstream of chr 11:
36572862;
(x) the first homology region is homologous to a region upstream of chr 11:
36571457
and the second homology region is homologous to a region downstream of chr 11:
36571460;
(xi) the first homology region is homologous to a region upstream of chr 11:
36569351
and the second homology region is homologous to a region downstream of chr 11:
36569354; or
(xii) the first homology region is homologous to a region upstream of chr 11:
36572375
and the second homology region is homologous to a region downstream of chr 11:
36572378.
In some embodiments:
(i) the first homology region is homologous to a region upstream of chr 11:
36569295
and the second homology region is homologous to a region downstream of chr 11:
36569298;
(ii) the first homology region is homologous to a region upstream of chr 11:
36573351
and the second homology region is homologous to a region downstream of chr 11:
36573354; or
(iii) the first homology region is homologous to a region upstream of chr 11:
36571366
and the second homology region is homologous to a region downstream of chr 11:
36571369.
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In some embodiments, the first homology region is homologous to a region
upstream of chr
11: 36569295 and the second homology region is homologous to a region
downstream of chr
11:36569298.
In some embodiments:
(i) the first homology region is homologous to a region comprising chr 11:
36569245-
36569294 and the second homology region is homologous to a region comprising
chr
11: 36569299-36569348;
(ii) the first homology region is homologous to a region comprising chr 11:
36573740-
36573789 and the second homology region is homologous to a region comprising
chr
11:36573794-36573843;
(iii) the first homology region is homologous to a region comprising chr 11:
36573591-
36573640 and the second homology region is homologous to a region comprising
chr
11: 36573645-36573694;
(iv) the first homology region is homologous to a region comprising chr 11:
36573301-
36573350 and the second homology region is homologous to a region comprising
chr
11: 36573355-36573404;
(v) the first homology region is homologous to a region comprising chr 11:
36569030-
36569079 and the second homology region is homologous to a region comprising
chr
11: 36569084-36569133;
(vi) the first homology region is homologous to a region comprising chr 11:
36572422-
36572471 and the second homology region is homologous to a region comprising
chr
11: 36572476-36572525;
(vii) the first homology region is homologous to a region comprising chr 11:
36571408-
36571457 and the second homology region is homologous to a region comprising
chr
11:36571462-36571511;
(viii) the first homology region is homologous to a region comprising chr 11:
36571316-
36571365 and the second homology region is homologous to a region comprising
chr
11: 36571370-36571419;
(ix) the first homology region is homologous to a region comprising chr 11:
36572809-
36572858 and the second homology region is homologous to a region comprising
chr
11: 36572863-36572912;
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(X) the first homology region is homologous to a region comprising chr
11:36571407-
36571456 and the second homology region is homologous to a region comprising
chr
11:36571461-36571510;
(xi) the first homology region is homologous to a region comprising chr 11:
36569301-
36569350 and the second homology region is homologous to a region comprising
chr
11:36569355-36569404; or
(xii) the first homology region is homologous to a region comprising chr 11:
36572325-
36572374 and the second homology region is homologous to a region comprising
chr
11: 36572379-36572428.
In some embodiments:
(i) the first homology region is homologous to a region comprising chr 11:
36569245-
36569294 and the second homology region is homologous to a region comprising
chr
11: 36569299-36569348;
(ii) the first homology region is homologous to a region comprising chr
11:36573301-
36573350 and the second homology region is homologous to a region comprising
chr
11:36573355-36573404; or
(iii) the first homology region is homologous to a region comprising chr 11:
36571316-
36571365 and the second homology region is homologous to a region comprising
chr
11: 36571370-36571419.
In some embodiments, the first homology region is homologous to a region
comprising chr 11:
36569245-36569294 and the second homology region is homologous to a region
comprising
chr 11: 36569299-36569348.
In some embodiments, the first homology region is homologous to a region
comprising chr 11:
36573740-36573789 and the second homology region is homologous to a region
comprising
chr 11: 36573794-36573843.
In some embodiments, the first homology region is homologous to a region
comprising chr 11:
36573591-36573640 and the second homology region is homologous to a region
comprising
chr 11: 36573645-36573694.
In some embodiments, the first homology region is homologous to a region
comprising chr 11:
36573301-36573350 and the second homology region is homologous to a region
comprising
chr 11: 36573355-36573404.
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In some embodiments, the first homology region is homologous to a region
comprising chr 11:
36569030-36569079 and the second homology region is homologous to a region
comprising
chr 11: 36569084-36569133.
In some embodiments, the first homology region is homologous to a region
comprising chr 11:
36572422-36572471 and the second homology region is homologous to a region
comprising
chr 11: 36572476-36572525.
In some embodiments, the first homology region is homologous to a region
comprising chr 11:
36571408-36571457 and the second homology region is homologous to a region
comprising
chr 11:36571462-36571511.
In some embodiments, the first homology region is homologous to a region
comprising chr 11:
36571316-36571365 and the second homology region is homologous to a region
comprising
chill: 36571370-36571419.
In some embodiments, the first homology region is homologous to a region
comprising chr 11:
36572809-36572858 and the second homology region is homologous to a region
comprising
chr 11: 36572863-36572912.
In some embodiments, the first homology region is homologous to a region
comprising chr 11:
36571407-36571456 and the second homology region is homologous to a region
comprising
chr 11: 36571461-36571510.
In some embodiments, the first homology region is homologous to a region
comprising chr 11:
36569301-36569350 and the second homology region is homologous to a region
comprising
chr 11: 36569355-36569404.
In some embodiments, the first homology region is homologous to a region
comprising chr 11:
36572325-36572374 and the second homology region is homologous to a region
comprising
chr 11: 36572379-36572428.
Exemplary homology regions are shown below in Table 1.
In some embodiments, the first homology region comprises or consists of a
nucleotide
sequence that has at least 70% identity, at least 80% identity, at least 90%
identity, at least
95% identity, at least 98% identity, or 100% identity to any of SEQ ID NOs: 7-
18 and/or the
second homology region comprises or consists of a nucleotide sequence that has
at least 70%
identity, at least 80% identity, at least 90% identity, at least 95% identity,
at least 98% identity,
or 100% identity to any of SEQ ID NOs: 19-30.
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Table 1 - Exemplary homology regions
Guide RNA First homology region Second homology region
9 TGCTGTGTGGAGGGAGGCACGC ATGGCAGTGGCCGGTGGGGACAG
CTGTAGCTCTGATGTCAGATGGC GGCTGAGCCAGCACCAACCACTCA
AATGT (SEQ ID NO: 7) GCC (SEQ ID NO: 19)
1 AAGAGAGCTACTTCCTGGCCGGA AAGGCAGATGTTGACTCGATCCAC
CCTCATTGCCAAGGTTTTCCGGA CCCACTGAGTTCTGCCATAACTGCT
TCGA (SEQ ID NO: 8) G (SEQ ID NO: 20)
2 AAGCAAGAGGCAAAGCGATCCAT GTGGGAATTCTTTTAGAGCTGATGA
CAAGCCAACCTTCGACATCTCTG GCACAACAGGAGATATCCAGTCCA
CCGC (SEQ ID NO: 9) T (SEQ ID NO: 21)
3 CAGCATGGCAGCCTCTTTCCCAC AATTCAGCACCCACATATTAAATTTT
CCACCTTGGGACTCAGTTCTGCC CAGAATGGAAATTTAAGCTGTTCC
CCAG (SEQ ID NO: 10) (SEQ ID NO: 22)
4 TTGTGTACAGACTAAGTTGAAGAT GGGGTGTATGTGTGTGGGTATAGG
GTTAGGAGGGAAGATTGTGGGCC GTGGGCAGCTGGGATGGAAATGGG
AAG (SEQ ID NO: 11) GG (SEQ ID NO: 23)
TTACTCCCACCTCTTCTTATTATG CAACAGTGACTTTCAGGATGACCTG
TTACAAACTATAGTGCTAATGACC TGTGAGTTTTATCTGAAACCATGTG
AT (SEQ ID NO: 12) (SEQ ID NO: 24)
6 ACAGAAGAGAATTAGGAAGCAGA TGGGCTGCAGTTGAAATATTTTTTG
ATTGAACTATAAGCAATTTTGAGG AGGTTAATGAGACATTTGAAATGGC
TGT (SEQ ID NO: 13) (SEQ ID NO: 25)
7 GGGAAGTAAAATGCTAAAGGAAT GAGGCAGGGGAGCCACAGGGAAA
GAGAAGGCATTTGGGGTTGAGTT GACCTAGCACCTGCCACAGAAGAG
CAAC (SEQ ID NO: 14) AAT (SEQ ID NO: 26)
8 AACCAACCCCCTGGAAGACTGCT TACAATGAGGCTAATACAATGTGGA
TTAAAAAGCTGGAAATACATTGTC AAATATTACTTTTCTTTGATTTTAG
CAG (SEQ ID NO: 15) (SEQ ID NO: 27)
CACAGAAGAGAATTAGGAAGCAG TTGGGCTGCAGTTGAAATATTTTTT
AATTGAACTATAAGCAATTTTGAG GAGGTTAATGAGACATTTGAAATGG
GTG (SEQ ID NO: 16) (SEQ ID NO: 28)
11 GGCAGTGGCCGGTGGGGACAGG ATCCCGAGGCTGGTCTACTGCTGA
GCTGAGCCAGCACCAACCACTCA GACCTTTTGTTAGAAGAGAGGAGAT
GCCTT (SEQ ID NO: 17) C (SEQ ID NO: 29)
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12
TTTTTTCTGCATCGCTAGCGATCT CTGGCATATGTAATTGCCAATGTTT
GTGCATTACAACTCAAATCAGTC TTTACCAGAAGAGAAACATTACTCC
GGG (SEQ ID NO: 18) (SEQ ID NO: 30)
Preferably, the first and second homology regions comprise or consist of
nucleotide
sequences that have at least 70% identity, at least 80% identity, at least 90%
identity, at least
95% identity, at least 98% identity, or 100% identity to first and second
homology regions in
the same row of Table 1. Suitably, the first homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90% identity,
at least 95% identity, at least 98% identity, or 100% identity to any of SEQ
ID NOs: 7-18 and
the second homology region comprises or consists of a nucleotide sequence that
has at least
70% identity, at least 80% identity, at least 90% identity, at least 95%
identity, at least 98%
identity, or 100% identity to the corresponding nucleotide sequence in Table 1
(i.e. SEQ ID
NOs: 19-30). For example, in some embodiments:
(i) the first homology region comprises or consists of a nucleotide sequence
that has
at least 70% identity, at least 80% identity, at least 90% identity, at least
95% identity,
at least 98% identity, or 100% identity to SEQ ID NO: 7 and the second
homology
region comprises or consists of a nucleotide sequence that has at least 70%
identity,
at least 80% identity, at least 90% identity, at least 95% identity, at least
98% identity,
or 100% identity to SEQ ID NO: 19;
(ii) the first homology region comprises or consists of a nucleotide sequence
that has
at least 70% identity, at least 80% identity, at least 90% identity, at least
95% identity,
at least 98% identity, or 100% identity to SEQ ID NO: 8 and the second
homology
region comprises or consists of a nucleotide sequence that has at least 70%
identity,
at least 80% identity, at least 90% identity, at least 95% identity, at least
98% identity,
or 100% identity to SEQ ID NO: 20;
(iii) the first homology region comprises or consists of a nucleotide sequence
that has
at least 70% identity, at least 80% identity, at least 90% identity, at least
95% identity,
at least 98% identity, or 100% identity to SEQ ID NO: 9 and the second
homology
region comprises or consists of a nucleotide sequence that has at least 70%
identity,
at least 80% identity, at least 90% identity, at least 95% identity, at least
98% identity,
or 100% identity to SEQ ID NO: 21;
(iv) the first homology region comprises or consists of a nucleotide sequence
that has
at least 70% identity, at least 80% identity, at least 90% identity, at least
95% identity,
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at least 98% identity, or 100% identity to SEQ ID NO: 10 and the second
homology
region comprises or consists of a nucleotide sequence that has at least 70%
identity,
at least 80% identity, at least 90% identity, at least 95% identity, at least
98% identity,
or 100% identity to SEQ ID NO: 22;
(v) the first homology region comprises or consists of a nucleotide sequence
that has
at least 70% identity, at least 80% identity, at least 90% identity, at least
95% identity,
at least 98% identity, or 100% identity to SEQ ID NO: 11 and the second
homology
region comprises or consists of a nucleotide sequence that has at least 70%
identity,
at least 80% identity, at least 90% identity, at least 95% identity, at least
98% identity,
or 100% identity to SEQ ID NO: 23;
(vi) the first homology region comprises or consists of a nucleotide sequence
that has
at least 70% identity, at least 80% identity, at least 90% identity, at least
95% identity,
at least 98% identity, or 100% identity to SEQ ID NO: 12 and the second
homology
region comprises or consists of a nucleotide sequence that has at least 70%
identity,
at least 80% identity, at least 90% identity, at least 95% identity, at least
98% identity,
or 100% identity to SEQ ID NO: 24;
(vii) the first homology region comprises or consists of a nucleotide sequence
that has
at least 70% identity, at least 80% identity, at least 90% identity, at least
95% identity,
at least 98% identity, or 100% identity to SEQ ID NO: 13 and the second
homology
region comprises or consists of a nucleotide sequence that has at least 70%
identity,
at least 80% identity, at least 90% identity, at least 95% identity, at least
98% identity,
or 100% identity to SEQ ID NO: 25;
(viii) the first homology region comprises or consists of a nucleotide
sequence that has
at least 70% identity, at least 80% identity, at least 90% identity, at least
95% identity,
at least 98% identity, or 100% identity to SEQ ID NO: 14 and the second
homology
region comprises or consists of a nucleotide sequence that has at least 70%
identity,
at least 80% identity, at least 90% identity, at least 95% identity, at least
98% identity,
or 100% identity to SEQ ID NO: 26;
(ix) the first homology region comprises or consists of a nucleotide sequence
that has
at least 70% identity, at least 80% identity, at least 90% identity, at least
95% identity,
at least 98% identity, or 100% identity to SEQ ID NO: 15 and the second
homology
region comprises or consists of a nucleotide sequence that has at least 70%
identity,
at least 80% identity, at least 90% identity, at least 95% identity, at least
98% identity,
or 100% identity to SEQ ID NO: 27;
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(X) the first homology region comprises or consists of a nucleotide sequence
that has
at least 70% identity, at least 80% identity, at least 90% identity, at least
95% identity,
at least 98% identity, or 100% identity to SEQ ID NO: 16 and the second
homology
region comprises or consists of a nucleotide sequence that has at least 70%
identity,
at least 80% identity, at least 90% identity, at least 95% identity, at least
98% identity,
or 100% identity to SEQ ID NO: 28;
(xi) the first homology region comprises or consists of a nucleotide sequence
that has
at least 70% identity, at least 80% identity, at least 90% identity, at least
95% identity,
at least 98% identity, or 100% identity to SEQ ID NO: 17 and the second
homology
region comprises or consists of a nucleotide sequence that has at least 70%
identity,
at least 80% identity, at least 90% identity, at least 95% identity, at least
98% identity,
or 100% identity to SEQ ID NO: 29; or
(xii) the first homology region comprises or consists of a nucleotide sequence
that has
at least 70% identity, at least 80% identity, at least 90% identity, at least
95% identity,
at least 98% identity, or 100% identity to SEQ ID NO: 18 and the second
homology
region comprises or consists of a nucleotide sequence that has at least 70%
identity,
at least 80% identity, at least 90% identity, at least 95% identity, at least
98% identity,
or 100% identity to SEQ ID NO: 30.
In some embodiments:
(i) the first homology region comprises or consists of a nucleotide sequence
that has
at least 70% identity, at least 80% identity, at least 90% identity, at least
95% identity,
at least 98% identity, or 100% identity to SEQ ID NO: 7 and the second
homology
region comprises or consists of a nucleotide sequence that has at least 70%
identity,
at least 80% identity, at least 90% identity, at least 95% identity, at least
98% identity,
or 100% identity to SEQ ID NO: 19;
(ii) the first homology region comprises or consists of a nucleotide sequence
that has
at least 70% identity, at least 80% identity, at least 90% identity, at least
95% identity,
at least 98% identity, or 100% identity to SEQ ID NO: 10 and the second
homology
region comprises or consists of a nucleotide sequence that has at least 70%
identity,
at least 80% identity, at least 90% identity, at least 95% identity, at least
98% identity,
or 100% identity to SEQ ID NO: 22; or
(iii) the first homology region comprises or consists of a nucleotide sequence
that has
at least 70% identity, at least 80% identity, at least 90% identity, at least
95% identity,
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at least 98% identity, or 100% identity to SEQ ID NO: 14 and the second
homology
region comprises or consists of a nucleotide sequence that has at least 70%
identity,
at least 80% identity, at least 90% identity, at least 95% identity, at least
98% identity,
or 100% identity to SEQ ID NO: 26.
In some embodiments, the first homology region comprises or consists of a
nucleotide
sequence that has at least 70% identity, at least 80% identity, at least 90%
identity, at least
95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 7 and the
second homology
region comprises or consists of a nucleotide sequence that has at least 70%
identity, at least
80% identity, at least 90% identity, at least 95% identity, at least 98%
identity, or 100% identity
to SEQ ID NO: 19.
In some embodiments, the first homology region comprises or consists of a
nucleotide
sequence that has at least 70% identity, at least 80% identity, at least 90%
identity, at least
95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 8 and the
second homology
region comprises or consists of a nucleotide sequence that has at least 70%
identity, at least
80% identity, at least 90% identity, at least 95% identity, at least 98%
identity, or 100% identity
to SEQ ID NO: 20.
In some embodiments, the first homology region comprises or consists of a
nucleotide
sequence that has at least 70% identity, at least 80% identity, at least 90%
identity, at least
95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 9 and the
second homology
region comprises or consists of a nucleotide sequence that has at least 70%
identity, at least
80% identity, at least 90% identity, at least 95% identity, at least 98%
identity, or 100% identity
to SEQ ID NO: 21.
In some embodiments, the first homology region comprises or consists of a
nucleotide
sequence that has at least 70% identity, at least 80% identity, at least 90%
identity, at least
95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 10 and the
second
homology region comprises or consists of a nucleotide sequence that has at
least 70%
identity, at least 80% identity, at least 90% identity, at least 95% identity,
at least 98% identity,
or 100% identity to SEQ ID NO: 22.
In some embodiments, the first homology region comprises or consists of a
nucleotide
sequence that has at least 70% identity, at least 80% identity, at least 90%
identity, at least
95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 11 and the
second
homology region comprises or consists of a nucleotide sequence that has at
least 70%
identity, at least 80% identity, at least 90% identity, at least 95% identity,
at least 98% identity,
or 100% identity to SEQ ID NO: 23.
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In some embodiments, the first homology region comprises or consists of a
nucleotide
sequence that has at least 70% identity, at least 80% identity, at least 90%
identity, at least
95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 12 and the
second
homology region comprises or consists of a nucleotide sequence that has at
least 70%
identity, at least 80% identity, at least 90% identity, at least 95% identity,
at least 98% identity,
or 100% identity to SEQ ID NO: 24.
In some embodiments, the first homology region comprises or consists of a
nucleotide
sequence that has at least 70% identity, at least 80% identity, at least 90%
identity, at least
95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 13 and the
second
homology region comprises or consists of a nucleotide sequence that has at
least 70%
identity, at least 80% identity, at least 90% identity, at least 95% identity,
at least 98% identity,
or 100% identity to SEQ ID NO: 25.
In some embodiments, the first homology region comprises or consists of a
nucleotide
sequence that has at least 70% identity, at least 80% identity, at least 90%
identity, at least
95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 14 and the
second
homology region comprises or consists of a nucleotide sequence that has at
least 70%
identity, at least 80% identity, at least 90% identity, at least 95% identity,
at least 98% identity,
or 100% identity to SEQ ID NO: 26.
In some embodiments, the first homology region comprises or consists of a
nucleotide
sequence that has at least 70% identity, at least 80% identity, at least 90%
identity, at least
95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 15 and the
second
homology region comprises or consists of a nucleotide sequence that has at
least 70%
identity, at least 80% identity, at least 90% identity, at least 95% identity,
at least 98% identity,
or 100% identity to SEQ ID NO: 27.
In some embodiments, the first homology region comprises or consists of a
nucleotide
sequence that has at least 70% identity, at least 80% identity, at least 90%
identity, at least
95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 16 and the
second
homology region comprises or consists of a nucleotide sequence that has at
least 70%
identity, at least 80% identity, at least 90% identity, at least 95% identity,
at least 98% identity,
or 100% identity to SEQ ID NO: 28.
In some embodiments, the first homology region comprises or consists of a
nucleotide
sequence that has at least 70% identity, at least 80% identity, at least 90%
identity, at least
95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 17 and the
second
homology region comprises or consists of a nucleotide sequence that has at
least 70%
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identity, at least 80% identity, at least 90% identity, at least 95% identity,
at least 98% identity,
or 100% identity to SEQ ID NO: 29.
In some embodiments, the first homology region comprises or consists of a
nucleotide
sequence that has at least 70% identity, at least 80% identity, at least 90%
identity, at least
95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 18 and the
second
homology region comprises or consists of a nucleotide sequence that has at
least 70%
identity, at least 80% identity, at least 90% identity, at least 95% identity,
at least 98% identity,
or 100% identity to SEQ ID NO: 30.
In some embodiments, the first homology region comprises or consists of a
nucleotide
sequence that has at least 98% identity to SEQ ID NO: 7 and the second
homology region
comprises or consists of a nucleotide sequence that has at least 98% identity
to SEQ ID NO:
19.
In some embodiments, the first homology region comprises or consists of the
nucleotide
sequence of SEQ ID NO: 7 and the second homology region comprises or consists
of the
nucleotide sequence of SEQ ID NO: 19.
In some embodiments, the 3' terminal sequence of the first homology region
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90% identity,
at least 95% identity, at least 98% identity, or 100% identity to any of SEQ
ID NOs: 7-18 and/or
the 5' terminal sequence of the second homology region comprises or consists
of a nucleotide
sequence that has at least 70% identity, at least 80% identity, at least 90%
identity, at least
95% identity, at least 98% identity, or 100% identity to any of SEQ ID NOs: 19-
30.
Suitably, the 3' terminal sequence of the first homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90% identity,
at least 95% identity, at least 98% identity, or 100% identity to any of SEQ
ID NOs: 7-18 and
the 5' terminal sequence of the second homology region comprises or consists
of a nucleotide
sequence that has at least 70% identity, at least 80% identity, at least 90%
identity, at least
95% identity, at least 98% identity, or 100% identity to the corresponding
nucleotide sequence
in Table 1 (i.e. SEQ ID NOs: 19-30).
For example, in some embodiments:
(i) the 3' terminal sequence of the first homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90%
identity, at least 95% identity, at least 98% identity, or 100% identity to
SEQ ID NO: 7
and the 5' terminal sequence of the second homology region comprises or
consists of
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a nucleotide sequence that has at least 70% identity, at least 80% identity,
at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID
NO: 19;
(ii) the 3' terminal sequence of the first homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90%
identity, at least 95% identity, at least 98% identity, or 100% identity to
SEQ ID NO: 8
and the 5' terminal sequence of the second homology region comprises or
consists of
a nucleotide sequence that has at least 70% identity, at least 80% identity,
at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID
NO: 20;
(iii) the 3' terminal sequence of the first homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90%
identity, at least 95% identity, at least 98% identity, or 100% identity to
SEQ ID NO: 9
and the 5' terminal sequence of the second homology region comprises or
consists of
a nucleotide sequence that has at least 70% identity, at least 80% identity,
at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID
NO: 21;
(iv) the 3' terminal sequence of the first homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90%
identity, at least 95% identity, at least 98% identity, or 100% identity to
SEQ ID NO: 10
and the 5' terminal sequence of the second homology region comprises or
consists of
a nucleotide sequence that has at least 70% identity, at least 80% identity,
at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID
NO: 22;
(v) the 3' terminal sequence of the first homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90%
identity, at least 95% identity, at least 98% identity, or 100% identity to
SEQ ID NO: 11
and the 5' terminal sequence of the second homology region comprises or
consists of
a nucleotide sequence that has at least 70% identity, at least 80% identity,
at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID
NO: 23;
(vi) the 3' terminal sequence of the first homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90%
identity, at least 95% identity, at least 98% identity, or 100% identity to
SEQ ID NO: 12
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and the 5' terminal sequence of the second homology region comprises or
consists of
a nucleotide sequence that has at least 70% identity, at least 80% identity,
at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID
NO: 24;
(vii) the 3' terminal sequence of the first homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90%
identity, at least 95% identity, at least 98% identity, or 100% identity to
SEQ ID NO: 13
and the 5' terminal sequence of the second homology region comprises or
consists of
a nucleotide sequence that has at least 70% identity, at least 80% identity,
at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID
NO: 25;
(viii) the 3' terminal sequence of the first homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90%
identity, at least 95% identity, at least 98% identity, or 100% identity to
SEQ ID NO: 14
and the 5' terminal sequence of the second homology region comprises or
consists of
a nucleotide sequence that has at least 70% identity, at least 80% identity,
at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID
NO: 26;
(ix) the 3' terminal sequence of the first homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90%
identity, at least 95% identity, at least 98% identity, or 100% identity to
SEQ ID NO: 15
and the 5' terminal sequence of the second homology region comprises or
consists of
a nucleotide sequence that has at least 70% identity, at least 80% identity,
at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID
NO: 27;
(x) the 3' terminal sequence of the first homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90%
identity, at least 95% identity, at least 98% identity, or 100% identity to
SEQ ID NO: 16
and the 5' terminal sequence of the second homology region comprises or
consists of
a nucleotide sequence that has at least 70% identity, at least 80% identity,
at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID
NO: 28;
(xi) the 3' terminal sequence of the first homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90%
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identity, at least 95% identity, at least 98% identity, or 100% identity to
SEQ ID NO: 17
and the 5' terminal sequence of the second homology region comprises or
consists of
a nucleotide sequence that has at least 70% identity, at least 80% identity,
at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID
NO: 29; or
(xii) the 3' terminal sequence of the first homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90%
identity, at least 95% identity, at least 98% identity, or 100% identity to
SEQ ID NO: 18
and the 5' terminal sequence of the second homology region comprises or
consists of
a nucleotide sequence that has at least 70% identity, at least 80% identity,
at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID
NO: 30.
In some embodiments:
(i) the 3' terminal sequence of the first homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90%
identity, at least 95% identity, at least 98% identity, or 100% identity to
SEQ ID NO: 7
and the 5' terminal sequence of the second homology region comprises or
consists of
a nucleotide sequence that has at least 70% identity, at least 80% identity,
at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID
NO: 19;
(ii) the 3' terminal sequence of the first homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90%
identity, at least 95% identity, at least 98% identity, or 100% identity to
SEQ ID NO: 10
and the 5' terminal sequence of the second homology region comprises or
consists of
a nucleotide sequence that has at least 70% identity, at least 80% identity,
at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID
NO: 22; or
(iii) the 3' terminal sequence of the first homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90%
identity, at least 95% identity, at least 98% identity, or 100% identity to
SEQ ID NO: 14
and the 5' terminal sequence of the second homology region comprises or
consists of
a nucleotide sequence that has at least 70% identity, at least 80% identity,
at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID
NO: 26.
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In some embodiments, the 3' terminal sequence of the first homology region
comprises or
consists of a nucleotide sequence that has at least 70% identity, at least 80%
identity, at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID NO: 7
and the 5' terminal sequence of the second homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90% identity,
at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO:
19.
In some embodiments, the 3' terminal sequence of the first homology region
comprises or
consists of a nucleotide sequence that has at least 70% identity, at least 80%
identity, at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID NO: 8
and the 5' terminal sequence of the second homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90% identity,
at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO:
20.
In some embodiments, the 3' terminal sequence of the first homology region
comprises or
consists of a nucleotide sequence that has at least 70% identity, at least 80%
identity, at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID NO: 9
and the 5' terminal sequence of the second homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90% identity,
at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO:
21.
In some embodiments, the 3' terminal sequence of the first homology region
comprises or
consists of a nucleotide sequence that has at least 70% identity, at least 80%
identity, at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID NO: 10
and the 5' terminal sequence of the second homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90% identity,
at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO:
22.
In some embodiments, the 3' terminal sequence of the first homology region
comprises or
consists of a nucleotide sequence that has at least 70% identity, at least 80%
identity, at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID NO: 11
and the 5' terminal sequence of the second homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least BO% identity, at
least 90% identity,
at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO:
23.
In some embodiments, the 3' terminal sequence of the first homology region
comprises or
consists of a nucleotide sequence that has at least 70% identity, at least 80%
identity, at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID NO: 12
and the 5' terminal sequence of the second homology region comprises or
consists of a
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nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90% identity,
at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO:
24.
In some embodiments, the 3' terminal sequence of the first homology region
comprises or
consists of a nucleotide sequence that has at least 70% identity, at least 80%
identity, at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID NO: 13
and the 5' terminal sequence of the second homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90% identity,
at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO:
25.
In some embodiments, the 3' terminal sequence of the first homology region
comprises or
consists of a nucleotide sequence that has at least 70% identity, at least 80%
identity, at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID NO: 14
and the 5' terminal sequence of the second homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90% identity,
at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO:
26.
In some embodiments, the 3' terminal sequence of the first homology region
comprises or
consists of a nucleotide sequence that has at least 70% identity, at least 80%
identity, at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID NO: 15
and the 5' terminal sequence of the second homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90% identity,
at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO:
27.
In some embodiments, the 3' terminal sequence of the first homology region
comprises or
consists of a nucleotide sequence that has at least 70% identity, at least 80%
identity, at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID NO: 16
and the 5' terminal sequence of the second homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90% identity,
at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO:
28.
In some embodiments, the 3' terminal sequence of the first homology region
comprises or
consists of a nucleotide sequence that has at least 70% identity, at least 80%
identity, at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID NO: 17
and the 5' terminal sequence of the second homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least BO% identity, at
least 90% identity,
at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO:
29.
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In some embodiments, the 3' terminal sequence of the first homology region
comprises or
consists of a nucleotide sequence that has at least 70% identity, at least 80%
identity, at least
90% identity, at least 95% identity, at least 98% identity, or 100% identity
to SEQ ID NO: 18
and the 5' terminal sequence of the second homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90% identity,
at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO:
30.
In some embodiments, the 3' terminal sequence of the first homology region
comprises or
consists of a nucleotide sequence that has at least 98% identity to SEQ ID NO:
7 and the 5'
terminal sequence of the second homology region comprises or consists of a
nucleotide
sequence that has at least 98% identity to SEQ ID NO: 19.
In some embodiments, the 3' terminal sequence of the first homology region
comprises or
consists of the nucleotide sequence of SEQ ID NO: 7 and the 5' terminal
sequence of the
second homology region comprises or consists of the nucleotide sequence of SEQ
ID NO: 19.
In some embodiments, the first homology region comprises or consists of a
nucleotide
sequence that has at least 70% identity, at least 80% identity, at least 90%
identity, at least
95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 31, or a
fragment thereof;
and the second homology region comprises or consists of a nucleotide sequence
that has at
least 70% identity, at least 80% identity, at least 90% identity, at least 95%
identity, at least
98% identity, 0r100% identity to SEQ ID NO: 32, or a fragment thereof.
Suitably, the fragments
are at least 50 bp in length, for example 50-250 bp or 100-200 bp in length.
In some embodiments, the first homology region comprises or consists of a
nucleotide
sequence that has at least 98% identity to SEQ ID NO: 31, or a fragment
thereof; and the
second homology region comprises or consists of a nucleotide sequence that has
at least 98%
identity to SEQ ID NO: 32, or a fragment thereof.
In some embodiments, the first homology region comprises or consists of the
nucleotide of
SEQ ID NO: 31, or a fragment thereof, and the second homology region comprises
or consists
of the nucleotide sequence of SEQ ID NO: 32, or a fragment thereof.
Illustrative first homology region for guide RNA 9 (SEQ ID NO: 31)
tgagcacacagttattacttggaaattgtgtacagactaagttgaagatgttaggagggaagattgtgggccaagtaac
ggggtgtatgtgtgtgggtatagggtgggcagctgggatggaaatggggggctgctgctgctgctgcaccctggcctc
ctgaactaatgatatcactcaccagaaactactgttcctgcactgtccaagccaccccaaactagtttgtcaaaatgaa
t
ctgtgctgtgtggagggaggcacgcctgtagctctgatgtcagatggcaatgt
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Illustrative second homology region for guide RNA 9 (SEQ ID NO: 32)
atggcagtggccggtggggacagggctgagccagcaccaaccactcagcctttgagatcccgaggctggtctactg
ctgagacclittgttagaagagaggagatcaagcatttgcaaggffictgagtgtcaaaatatgaatccaagataactc
tt
tcacaatcctaacttcatgctgtctacaggtccatattttagcctgctttctccatgttcatccgaaaagaaagaaaag
cta
agggtggtggtcatatttgaaattagccagatcttaagttffictgggggaaatttagaagaaaatatggaaaagtgac
ta
tgagcaca
Genome insertion sites
The site of the double-strand break (DSB) can be introduced specifically by
any suitable
technique, for example using a CRISPR/Cas9 system and the guide RNAs disclosed
herein.
In the present invention, the DSB is introduced into the RAG1 intron 1 or RAG1
exon 2. For
example, a DSB may be introduced at any of the sites recited in Table 2 below.
Optionally, a
DSB is introduced into the RAG1 intron 1.
Table 2¨ Exemplary DSB sites in RAG1 intron 1 or RAG1 exon 2
Guide Exemplary DSB site
9 between chr 11: 36569296 and 36569297
1 between chr 11: 36573791 and 36573792
2 between chr 11: 36573642 and 36573643
3 between chr 11: 36573352 and 36573353
4 between chr 11: 36569081 and 36569082
5 between chr 11: 36572473 and 36572474
6 between chr 11:36571459 and 36571460
7 between chr 11: 36571367 and 36571368
8 between chr 11: 36572860 and 36572861
10 between chr 11:36571458 and 36571459
11 between chr 11: 36569352 and 36569353
12 between chr 11:36572376 and 36572377
Suitably, each homology region is homologous to a fragment of the RAG1 intron
1 and/or
RAG1 exon 2 either side of the DSB. For example, the first homology region may
be
homologous to a region in the RAG1 intron 1 and/or RAG1 exon 2 upstream of the
DSB and
the second homology region may be homologous to a region downstream of the
DSB.
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In the present invention, the nucleotide sequence insert (e.g. a splice
acceptor sequence and
a nucleotide sequence encoding a RAG1 polypeptide) may be introduced at the
DSB site by
homology-directed repair (HDR). Thus, the nucleotide insert (e.g. a splice
acceptor sequence
and a nucleotide sequence encoding a RAG1 polypeptide) may replace the region
of the
genome flanked by the homology regions and comprising the DSB.
As used herein, the "nucleotide sequence insert" may consist of the region of
the
polynucleotide flanked by the first homology region and the second homology
region. For
example, the nucleotide sequence insert may comprise a splice acceptor
sequence and a
nucleotide sequence encoding a RAG1 polypeptide.
The nucleotide sequence insert may be introduced into a genome at any of the
sites recited
in Table 2 above. In other words, the genome of the present invention may
comprise the
nucleotide sequence insert at any of the sites recited in Table 2 above.
In some embodiments, the nucleotide sequence insert is introduced:
(i) between chr 11: 36569296 and 36569297;
(ii) between chr 11: 36573352 and 36573353; or
(iii) between chr 11: 36571367 and 36571368.
In some embodiments, the nucleotide sequence insert is introduced between chr
11:
36569296 and 36569297.
In some embodiments, the genome of the present invention comprises a
nucleotide sequence
comprising a splice acceptor sequence and a nucleotide sequence encoding a
RAG1
polypeptide, which is introduced:
(i) between chr 11: 36569296 and 36569297;
(ii) between chr 11: 36573352 and 36573353; or
(iii) between chr 11: 36571367 and 36571368.
In some embodiments, the genome of the present invention comprises a
nucleotide sequence
comprising a splice acceptor sequence and a nucleotide sequence encoding a
RAG1
polypeptide, which is introduced between chr 11: 36569296 and 36569297.
The nucleotide sequence insert may replace any of the regions recited in Table
3 below. In
other words, the genome of the present invention may comprise the nucleotide
sequence
insert replacing any of the regions recited in Table 3.
Table 3¨ Exemplary insertion sites in RAG1 intron 1 or RAG1 exon 2
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Guide Exemplary region to replace
9 chr 11: 36569295 to 36569298
1 chr 11: 36573790 to 36573793
2 chr 11: 36573641 to 36573644
3 chr 11: 36573351 to 36573354
4 chr 11: 36569080 to 36569083
chr 11: 36572472 to 36572475
6 chr 11: 36571458 to 36571461
7 chr 11: 36571366 to 36571369
8 chr 11: 36572859 to 36572862
chr 11: 36571457 to 36571460
11 chr 11: 36569351 to 36569354
12 chr 11: 36572375 to 36572378
In some embodiments, the nucleotide sequence insert replaces:
(i) chr 11: 36569295 to 36569298;
(ii) chr 11: 36573351 to 36573354; or
5 (iii) chr 11: 36571366 to 36571369.
In some embodiments, the nucleotide sequence insert replaces chr 11. 36569295
to
36569298.
In some embodiments, the genome of the present invention comprises a
nucleotide sequence
comprising a splice acceptor sequence and a nucleotide sequence encoding a
RAG1
10 polypeptide, which replaces:
(i) chr 11: 36569295 to 36569298;
(ii) chr 11: 36573351 to 36573354; or
(iii) chr 11: 36571366 to 36571369.
In some embodiments, the genome of the present invention comprises a
nucleotide sequence
comprising a splice acceptor sequence and a nucleotide sequence encoding a
RAG1
polypeptide, which replaces chr 11: 36569295 to 36569298.
Splice acceptor and donor sequences
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RNA splicing is a form of RNA processing in which a newly made precursor
messenger RNA
(pre-mRNA) transcript is transformed into a mature messenger RNA (mRNA).
During splicing,
introns (non-coding regions) are removed and exons (coding regions) are joined
together.
Within introns, a donor site (5 end of the intron), a branch site (near the 3
end of the intron)
and an acceptor site (3' end of the intron) are required for splicing. The
splice donor site
includes an almost invariant sequence GU at the 5' end of the intron, within a
larger, less
highly conserved region. The splice acceptor site at the 3' end of the intron
terminates the
intron with an almost invariant AG sequence. Upstream (5'-ward) from the AG
there is a region
high in pyrimidines (C and U), or polypyrimidine tract. Further upstream from
the
polypyrimidine tract is the branchpoint.
A "splice acceptor sequence" is a nucleotide sequence which can function as an
acceptor site
at the 3' end of the intron. Consensus sequences and frequencies of human
splice site regions
are described in Ma, S.L., et al., 2015. PLoS One, 10(6), p.e0130729.
Suitably, the splice acceptor sequence may comprise the nucleotide sequence
(Y)nNYAG,
where n is 10-20, or a variant with at least 90% or at least 95% sequence
identity. Suitably,
the splice acceptor sequence may comprise the sequence (Y)NCAG, where n is 10-
20, or a
variant with at least 90% or at least 95% sequence identity.
In some embodiments of the invention, the splice acceptor sequence comprises
or consists of
a nucleotide sequence which is at least 70% identical to SEQ ID NO: 33 or a
fragment thereof.
Suitably, the splice acceptor sequence comprises or consists of a nucleotide
sequence which
is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at
least 99% identical
to SEQ ID NO: 33 or a fragment thereof.
In some embodiments of the invention, the splice acceptor sequence comprises
or consists of
the nucleotide sequence SEQ ID NO: 33 or a fragment thereof.
Exemplary splice acceptor sequence (SEQ ID NO: 33)
ctgacctcttctcttcctcccacag
The polynucleotide of the invention may comprise a splice donor sequence. The
genome may
comprise a splice donor sequence in the RAG1 intron 1. Suitably, the splice
donor sequence
nucleotide sequence is 3' of the nucleotide sequence encoding a RAG1
polypeptide. The
splice donor sequence may be used to provide an mRNA comprising the RAG1
polypeptide
and RAG1 exon 2.
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A "splice donor sequence" is a nucleotide sequence which can function as a
donor site at the
5' end of the intron. Consensus sequences and frequencies of human splice site
regions are
describe in Ma, S.L., et al., 2015. PLoS One, 10(6), p.e0130729.
In some embodiments of the invention, the splice donor sequence comprises or
consists of a
nucleotide sequence which is at least 85% identical to SEQ ID NO: 34 or a
fragment thereof.
In some embodiments of the invention, the splice donor sequence comprises or
consists of
the nucleotide sequence SEQ ID NO: 34 or a fragment thereof.
Exemplary splice donor sequence (SEQ ID NO: 34)
aggtaagt
In some embodiments of the invention, the polynucleotide of the invention does
not comprise
a splice donor sequence.
Regulatory elements
The polynucleotide of the invention may comprise one or more regulatory
elements which may
act pre- or post-transcriptionally. Suitably, the nucleotide sequence encoding
a RAG1
polypeptide is operably linked to one or more regulatory elements which may
act pre- or post-
transcriptionally. The one or more regulatory elements may facilitate
expression of the RAG1
polypeptide in the cells of the invention.
A "regulatory element" is any nucleotide sequence which facilitates expression
of a
polypeptide, e.g. acts to increase expression of a transcript or to enhance
mRNA stability.
Suitable regulatory elements include for example promoters, enhancer elements,
post-
transcriptional regulatory elements and polyadenylation sites.
Polvadenvlation sequence
The polynucleotide of the invention may comprise a polyadenylation sequence.
Suitably, the
nucleotide sequence encoding a RAG1 polypeptide is operably linked to a
polyadenylation
sequence. The polyadenylation sequence may improve gene expression.
Suitable polyadenylation sequences will be well known to those of skill in the
art. Suitable
polyadenylation sequences include a bovine growth hormone (BGH)
polyadenylation
sequence or an early SV40 polyadenylation signal. In some embodiments of the
invention, the
polyadenylation sequence is a BGH polyadenylation sequence.
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In some embodiments of the invention, the polyadenylation sequence comprises
or consists
of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 35, 62
or 65 or a
fragment thereof. Suitably, the polyadenylation sequence comprises or consists
of a
nucleotide sequence which is at least 80%, at least 85%, at least 90%, at
least 95%, at least
98% or at least 99% identical to SEQ ID NO: 35, 62 01 65 or a fragment
thereof.
In some embodiments of the invention, the polyadenylation sequence comprises
or consists
of the nucleotide sequence SEQ ID NO: 35, 62 or 65 or a fragment thereof.
Exemplary BGH polyadenylation sequence (SEQ ID NO: 35)
Gctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactccca
ctgt
cctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcag
ga
cagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgg
Exemplary BGH polyadenylation sequence (SEQ ID NO: 62)
Actgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactccca
ctgc
cctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcag
ga
cagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgg
Exemplary BGH polyadenylation sequence (SEQ ID NO: 65)
ctgtgccttctagttgccagccatctgligtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccac
tgtc
ctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcagg
ac
agcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgg
Kozak sequence
The polynucleotide of the invention may comprise a Kozak sequence. Suitably,
the nucleotide
sequence encoding a RAG1 polypeptide is operably linked to a Kozak sequence. A
Kozak
sequence may be inserted before the start codon of the RAG1 polypeptide to
improve the
initiation of translation.
Suitable Kozak sequences will be well known to those of skill in the art.
In some embodiments of the invention, the Kozak sequence comprises or consists
of a
nucleotide sequence which is at least 70% identical to SEQ ID NO: 36 or a
fragment thereof.
Suitably, the Kozak sequence comprises or consists of a nucleotide sequence
which is at least
80%, or at least 90% identical to SEQ ID NO: 36 or a fragment thereof.
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In some embodiments of the invention, the Kozak sequence comprises or consists
of the
nucleotide sequence SEQ ID NO: 36 or a fragment thereof.
Exemplary Kozak sequence (SEQ ID NO: 36)
gccgccaccatg
Post-transcriptional reaulatory elements
The polynucleotide of the invention may comprise a post-transcriptional
regulatory element.
Suitably, the nucleotide sequence encoding a RAG1 polypeptide is operably
linked to a post-
transcriptional regulatory element. The post-transcriptional regulatory
element may improve
gene expression.
Suitable post-transcriptional regulatory elements will be well known to those
of skill in the art.
The polynucleotide of the invention may comprise a Woodchuck Hepatitis Virus
Post-
transcriptional Regulatory Element (WPRE). Suitably, the nucleotide sequence
encoding a
RAG1 polypeptide is operably linked to a WPRE.
In some embodiments of the invention, the WPRE comprises or consists of a
nucleotide
sequence which is at least 70% identical to SEQ ID NO: 37 or a fragment
thereof. Suitably,
the WPRE comprises or consists of a nucleotide sequence which is at least 80%,
or at least
90% identical to SEQ ID NO: 37 or a fragment thereof.
In some embodiments of the invention, the WPRE comprises or consists of the
nucleotide
sequence SEQ ID NO: 37 or a fragment thereof.
Exemplary WPRE (SEQ ID NO: 37)
aatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggat
acgct
gctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgt
ctctttatgag
gagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattg
c
caccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgcctt
gcc
cgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggc
tg
ctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttc
ctt
cccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggc
c
gcctccccgcctg
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In some embodiments of the invention, the RAG1 polypeptide is not operably
linked to a post-
transcriptional regulatory element. In some embodiments of the invention, the
RAG1
polypeptide is not operably linked to a WPRE.
Endogenous 3'UTR
The polynucleotide of the invention may comprise an endogenous RAG1 3'UTR.
Suitably, the
nucleotide sequence encoding a RAG1 polypeptide is operably linked to an
endogenous
RAG1 3'UTR.
In some embodiments of the invention, the RAG1 3'UTR comprises or consists of
a nucleotide
sequence which is at least 70% identical to SEQ ID NO: 38 or a fragment
thereof. Suitably,
the RAG1 3'UTR comprises or consists of a nucleotide sequence which is at
least 80%, or at
least 90% identical to SEQ ID NO: 38 or a fragment thereof.
In some embodiments of the invention, the RAG1 3'UTR comprises or consists of
the
nucleotide sequence SEQ ID NO: 38 or a fragment thereof.
Exemplaty RAG1 3'UTR (SEQ ID NO: 38)
gtagggcaaccacttatgagttggtttttgcaattgagtttccctctgggttgcattgagggcttctcctagcaccctt
tactg
ctgtgtatggggcttcaccatccaagaggtggtaggttggagtaagatgctacagatgctctcaagtcaggaatagaa
actgatgagctgattgcttgaggcttttagtgagttccgaaaagcaacaggaaaaatcagttatctgaaagctcagtaa
ctcagaacaggagtaactgcaggggaccagagatgagcaaagatctgtgtgtgttggggagctgtcatgtaaatcaa
agccaaggttgtcaaagaacagccagtgaggccaggaaagaaattggtcttgtggttlicattttlitcccccttgatt
gat
tatattttgtattgagatatgataagtgccttctatttcatttttgaataattcttcatttttataattttacatatct
tggcttgctatat
aagattcaaaagagctttttaaatttlictaataatatcttacatttgtacagcatgatgacctttacaaagtgctctc
aatgc
atttacccattcgttatataaatatgttacatcaggacaactttgagaaaatcagtcctthttatgtttaaattatgta
tctattgt
aaccttcagagtttaggaggtcatctgctgtcatggatttttcaataatgaatttagaatacacctgttagctacagtt
agtta
ttaaatcttctgataatatatgtttacttagctatcagaagccaagtatgattctttatttttactifttcatttcaag
aaatttagag
tttccaaatttagagcttctgcatacagtcttaaagccacagaggcttgtaaaaatataggttagcttgatgtctaaaa
ata
tatttcatgtcttactgaaacattttgccagactttctccaaatgaaacctgaatcaatttttctaaatctaggtttca
tagagtc
ctctcctctgcaatgtgttattctttctataatgatcagtttactttcagtggattcagaattgtgtagcaggataacc
ttgtatttt
tccatccgctaagtttagatggagtccaaacgcagtacagcagaagagttaacatttacacagtgctttttaccactgt
g
gaatgttttcacactcatttttccttacaacaattctgaggagtaggtgttgttattatctccatttgatgggggttta
aatgattt
gctcaaagtcatttaggggtaataaatacttggcttggaaatttaacacagtccttligtctccaaagcccttcttcli
tccac
cacaaattaatcactatgtttataaggtagtatcagaatttttttaggattcacaactaatcactatagcacatgacct
tggg
attacatlittatggggcaggggtaagcaagtlittaaatcatttgtgtgctctggctctlitgatagaagaaagcaac
acaa
aagctccaaagggccccctaaccctcttgtggctccagttatttggaaactatgatctgcatccttaggaatctgggat
ttg
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ccagttgctggcaatgtagagcaggcatggaattttatatgctagtgagtcataatgatatgttagtgliaattagttl
ittctt
cctttgattttattggccataattgctactcttcatacacagtatatcaaagagcttgataatttagttgtcaaaagtg
catcg
gcgacattatctttaattgtatgtatttggtgcttcttcagggattgaactcagtatctttcattaaaaaacacagcag
ttttcct
tgctttttatatgcagaatatcaaagtcatttctaatttagttgtcaaaaacatatacatattttaacattagtthttt
gaaaactc
ttggttttgtttttttggaaatgagtgggccactaagccacactttcccttcatcctgcttaatccttccagcatgtct
ctgcact
aataaacagctaaattcacataatcatcctatttactgaagcatggtcatgctggtttatagattttttacccatttct
actctttt
tctctattggtggcactgtaaatactttccagtattaaattatcctffictaacactgtaggaactattttgaatgcat
gtgacta
agagcatgatttatagcacaacctliccaataatcccttaatcagatcacattttgataaaccctgggaacatctggct
gc
aggaatttcaatatgtagaaacgctgcctatggttlittgcccttactgttgagactgcaatatcctagaccctagttt
tatact
agagttttatttttagcaatgcctattgcaagtgcaattatatactccagggaaattcaccacactgaatcgagcattt
gtgt
gtgtatgtgtgaagtatatactgggacttcagaagtgcaatgtatttttctcctgtgaaacctgaatctacaagttttc
ctgcc
aagccactcaggtgcattgcagggaccagtgataatggctgatgaaaattgatgattggtcagtgaggtcaaaagga
gccttgggattaataaacatgcactgagaagcaagaggaggagaaaaagatgtctttttcttccaggtgaactggaatt
tagttttgcctcagatttttttcccacaagatacagaagaagataaagatttttttggttgagagtgtgggtcttgcat
tacatc
aaacagagttcaaattccacacagataagaggcaggatatataagcgccagtggtagttgggaggaataaaccatt
atttggatgcaggtggtttttgattgcaaatatgtgtgtgtcttcagtgattgtatgacagatgatgtattcttttgat
gttaaaag
attttaagtaagagtagatacattgtacccattttacattttcttattttaactacagtaatctacataaatatacctc
agaaat
catttttggtgattattttttgttttgtagaattgcacttcagtttattttcttacaaataaccttacattttgtttaa
tggcttccaaga
gcctttlittlitttgtatttcagagaaaattcaggtaccaggatgcaatggatttatttgattcaggggacctgtgli
tccatgtc
aaatgttttcaaataaaatgaaatatgagtttcaatactttttatattttaatatttccattcattaatattatggtta
ttgtcagca
attttatgtttgaatatttgaaataaaagtttaagatttgaaaatggtatgtattataatttctattcaaatattaata
ataatattg
agtgcagcatt
In some embodiments of the invention, the RAG1 polypeptide is not operably
linked to a RAG1
3'UTR.
Further codinq sequences
The polynucleotide of the invention may comprise a further coding sequence.
The
polynucleotide of the invention may comprise an internal ribosome entry site
sequence (IRES).
The IRES may increase or allow expression of the further coding sequence. The
IRES may
be operably linked to the further coding sequence.
In some embodiments of the invention, the IRES comprises or consists of a
nucleotide
sequence which is at least 70% identical to SEQ ID NO: 63 or a fragment
thereof. Suitably,
the IRES comprises or consists of a nucleotide sequence which is at least 80%,
or at least
90% identical to SEQ ID NO: 63 or a fragment thereof.
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In some embodiments of the invention, the IRES comprises or consists of the
nucleotide
sequence SEQ ID NO: 63 or a fragment thereof.
Exemplary IRES (SEQ ID NO: 63)
gaattaactcgaggaattccgCccctctcccteccccccccctaacgttactggccgaagccgcttggaataaggccg
gtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtc
ttcttg
acgagcattctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctct
g
gaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcc
tctgcggccaaaagccaacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggat
agttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccatt
gtatgggatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaacgtctaggccccccg
a
accacggggacgtggttttcctttgaaaaacacgatgataatatggccacaacc
The further coding sequence may encode a selector, for example a NGFR
receptor, e.g. a low
affinity NGFR, such as a C-terminal truncated low affinity NGFR. The selector
may be used
for enrichment of cells.
In some embodiments of the invention, the NGFR-encoding sequence comprises or
consists
of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 64 or a
fragment
thereof. Suitably, the NGFR-encoding sequence comprises or consists of a
nucleotide
sequence which is at least 80%, or at least 90% identical to SEQ ID NO: 64 or
a fragment
thereof.
In some embodiments of the invention, the NGFR-encoding sequence comprises or
consists
of the nucleotide sequence SEQ ID NO: 64 or a fragment thereof.
Exemplary NGFR-encoding sequence (SEQ ID NO: 64)
atgggagctggtgctaccggcagagctatggatggacctagactgctgctcctgctgctgctcggagtttctcttggcg
g
agccaaagaggcctgtcctaccggcctgtatacacactctggcgagtgctgcaaggcctgcaatcttggagaaggcg
tggcacagccttgcggcgctaatcagacagtgtgcgagccttgcctggacagcgtgacctttagcgacgtggtgtctgc
caccgagccatgcaagccttgtaccgagtgtgtgggcctgcagagcatgtctgccccttgtgtggaagccgacgatgc
cgtgtgtagatgcgcctacggctactaccaggacgagacaacaggcagatgcgaggcctgtagagtgtgtgaagcc
ggctctggactggtgttcagctgccaagacaagcagaacaccgtgtgcgaggaatgccccgatggcacctatagcg
acgaggccaaccatgtagatccctgcctgccttgtactgtgtgcgaagataccgagcggcagctgcgcgagtgtaca
agatgggctgatgccgagtgcgaagagatccccggcagatggatcaccagaagcacacctccagagggcagcg
atagcacagccccttctacacaagagcccgaggctcctcctgagcaggatctgattgcctctacagtggccggcgtgg
tcacaacagtgatgggatcttctcagcccgtggtcaccagaggcaccaccgacaatctgatccccgtgtactgtagca
tcctggccgccgtggttgtgggactcgtggcctatatcgccttcaagcggtggaaccggggcatcctgtaa
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The further coding sequence may encode a destabilisation domain, for example a
peptide
sequence rich in proline (P), glutamic acid (E), serine (S), and threonine (T)
(PEST).
Endogenous RAG1 protein may be destabilized by the destabilisation domain,
e.g. PEST
signal peptide via proteasome degradation.
In some embodiments of the invention, the PEST-encoding sequence comprises or
consists
of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 66 or a
fragment
thereof. Suitably, the PEST-encoding sequence comprises or consists of a
nucleotide
sequence which is at least 80%, or at least 90% identical to SEQ ID NO: 66 or
a fragment
thereof.
In some embodiments of the invention, the PEST-encoding sequence comprises or
consists
of the nucleotide sequence SEQ ID NO: 66 or a fragment thereof.
Exemplary PEST-encoding sequence (SEQ ID NO: 66)
atgaggaccgaggcccccgagggcaccgagagcgagatggagacccccagcgccatcaacggcaaccccagc
tggcac
Promoters and enhancers
Suitably, the nucleotide sequence encoding a RAG1 polypeptide is operably
linked to a
promoter and/or enhancer element.
A "promoter" is a region of DNA that leads to initiation of transcription of a
gene. Promoters
are located near the transcription start sites of genes, upstream on the DNA
(towards the 5'
region of the sense strand). Any suitable promoter may be used, the selection
of which may
be readily made by the skilled person.
An "enhancer" is a region of DNA that can be bound by proteins (activators) to
increase the
likelihood that transcription of a particular gene will occur. Enhancers are
cis-acting. They can
be located up to 1 Mbp (1,000,000 bp) away from the gene, upstream or
downstream from the
start site. Any suitable enhancer may be used, the selection of which may be
readily made by
the skilled person.
Transcription of the nucleotide sequence encoding a RAG1 polypeptide may be
driven by an
endogenous promoter. For example, if the polynucleotide of the present
invention is inserted
into the RAG1 intron 1, transcription of the nucleotide sequence encoding a
RAG1 polypeptide
may be driven by the endogenous RAG1 promoter.
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In some embodiments of the invention, the polynucleotide of the invention does
not comprise
a promoter and/or enhancer element. In some embodiments of the invention, the
genome of
the invention does not comprise a promoter and/or enhancer element (e.g. an
exogenous
promoter and/or enhancer element) in the RAG1 intron 1.
Exemplary polynucleotides and genomes
In some embodiments, the polynucleotide of the invention comprises,
essentially consists of,
or consists of from 5' to 3': a first homology region, a splice acceptor
sequence, a nucleotide
sequence encoding a RAG1 polypeptide, a polyadenylation sequence and a second
homology
region.
In some embodiments, the polynucleotide of the invention comprises,
essentially consists of,
or consists of from 5' to 3': a first homology region, a splice acceptor
sequence, a kozak
sequence, a nucleotide sequence encoding a RAG1 polypeptide, a polyadenylation
sequence
and a second homology region.
In some embodiments, the polynucleotide of the invention comprises,
essentially consists of,
or consists of from 5' to 3': a first homology region, a splice acceptor
sequence, a nucleotide
sequence encoding a RAG1 polypeptide, a WPRE, a polyadenylation sequence and a
second
homology region.
In some embodiments, the polynucleotide of the invention comprises,
essentially consists of,
or consists of from 5' to 3': a first homology region, a splice acceptor
sequence, a kozak
sequence, a nucleotide sequence encoding a RAG1 polypeptide, a WPRE, a
polyadenylation
sequence and a second homology region.
In some embodiments, the polynucleotide of the invention comprises,
essentially consists of,
or consists of from 5' to 3': a first homology region, a splice acceptor
sequence, a kozak
sequence, a nucleotide sequence encoding a RAG1 polypeptide, a 3' UTR, a
polyadenylation
sequence and a second homology region.
In some embodiments, the polynucleotide of the invention comprises,
essentially consists of,
or consists of from 5' to 3': a first homology region, a splice acceptor
sequence, a kozak
sequence, a nucleotide sequence encoding a RAG1 polypeptide, an IRES, a
nucleotide
sequence encoding a selector (e.g. NGFR), a polyadenylation sequence and a
second
homology region.
In some embodiments, the polynucleotide of the invention comprises,
essentially consists of,
or consists of from 5' to 3': a first homology region, a splice acceptor
sequence, a kozak
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sequence, a nucleotide sequence encoding a RAG1 polypeptide, an IRES, a
nucleotide
sequence encoding a destabilisation domain (e.g. a PEST sequence), a splice
donor
sequence, and a second homology region.
In some embodiments, the polynucleotide of the invention comprises,
essentially consists of,
or consists of from 5' to 3': a first homology region, a splice acceptor
sequence, a nucleotide
sequence encoding a RAG1 polypeptide, a splice donor sequence and a second
homology
region.
In some embodiments, the polynucleotide of the invention comprises or consists
of a
nucleotide sequence that has at least 70%, at least 75%, at least 80%, at
least 85%, at least
90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 39.
In some embodiments, the polynucleotide of the invention comprises or consists
of the
nucleotide sequence of SEQ ID NO: 39.
In some embodiments, the genome of the invention comprises a nucleotide
sequence that has
at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, at least
98%, or at least 99% identity to SEQ ID NO: 39.
In some embodiments, the genome of the invention comprises the nucleotide
sequence of
SEQ ID NO: 39.
In some embodiments, the genome of the invention comprises a nucleotide
sequence that has
at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, at least
98%, or at least 99% identity to nucleotides 297-3687 of SEQ ID NO: 39 or
nucleotides 291-
3693 of SEQ ID NO: 39.
In some embodiments, the genome of the invention comprises the nucleotide
sequence of
nucleotides 297-3687 of SEQ ID NO: 39 or nucleotides 291-3693 of SEQ ID NO:
39.
Exemplary polynucleotide (SEQ ID NO: 39)
tgagcacacagttattacttggaaattgtgtacagactaagttgaagatgttaggagggaagattgtgggccaagtaac
ggggtgtatgtgtgtgggtatagggtgggcagctgggatggaaatggggggctgctgctgctgctgcaccctggcctc
ctgaactaatgatatcactcaccagaaactactgttcctgcactgtccaagccaccccaaactagtligtcaaaatgaa
t
ctgtgctgtgtggagggaggcacgcctgtagctctgatgtcagatggcaatgtgaattcctgacctcttctcttcctcc
cac
a g g ccg ccaccatg g ccg cctccttccca ccta cccttg g a ttg tcctccg cccctg a cg a
a attca acatcccca cat
caaattctcggagtggaagttcaagctctttcgcgtgcgctcgttcgaaaagacccccgaggaagcccaaaaggag
aagaaagactcattcgaaggaaaacccagcctcgaacagtccccggccgtcctggacaaggccgacgggcaga
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TT -17 -Z0Z 99Z5610
69
oo66e000meebleopeoT466600poebeeopoo6oeeleoe36oee6leon6ee6e3opoelBeeoopo
elelblobbpeoleobeebloblblebeebbleeeboeloblbeeoolbeoob000boeeblebeeebeillbboo
boollbnbeeoee366634beboeeobbbe6331336661336666owoollbbleboboeebolellebe6333
gc
blboemo boppebeeoeompepeemepebeeebbeeboelebeombeeoubeemebolbp bp beb
eobopbbeeeoobeoeemobeoelbeo1616pbolbebb000blbebbeeoobpooblbolobebbobbp16
1336ee6re6ee6poe464aoe664e343ee6363613636eebleoe6oee6ee6o6eamole6pee646134
66363e63),663eeebbeepebwbp6eebbep6olpeee66oeeb),e66364elle0006eebpoee6w
6ee6eeoboo400eoeeeoe611333e1366e3661663beee6eeebee66eeoopo6oee6000ee6eeo
0
e4646ee566634e6e64436e33ll34e6ee3e3p6e63363363ee3bb34e3eb36ne3op3363e6o4e3
4346O44446444466 6433
Te000eepp6obbpi6be6Tep6oeebbpoeebebbo6oe000lb6epeoTebopeoop6T6TpTee6eo
eal6o6BebBloBB000emBoe6361oppeobpleoepT600p66eopobeeBbpo666e6616ee636
36463136eeeeboebiepbbooeebbbboopoleonbeempebboblmelbbobbblobebbloblebpee
gz
6004o4bee64e3o66e666e6e66-3634e64po6o46p44eo3600ebpooeee6oe34e6o6e6e6oe6
63661164e61306161363o6ee3613513136e6eopee6o35ee3366e6ee6311646eee3463ee6e333
pbeleobobwooeowbeebwoleooe34331343636463366eeee6633646646133e663344663e3
6ee6e66316163e6o666lee661e63616o4ee66ee31664661663e3113336663ee3433epe63e66
pleBeeoeole6e6lee666e66poleoe66e66e66Teoe661e6poo66316166136363311e6oele6e
OZ
op6boeeeooboTeooeTebblbpooepebTebbT6ppopoTbpobbobeblobbbTebpelleobbbTboe
boaeombopolblboeeeeebn000boo beabbleebonleolemelebbboo bp 6113165eeee6ea boe
ebbolpooboeoopeoobeouplebeobboebbooemeoobbee616pebbeomblebeeoeoomeeoo
op6ieoppopeoeoeeolbobomeoobolo1616161obboobeoopebbbolebbeeeobbbeobleoleoo
66e6643ee64e6136ee3e6e3eoee63ee66313666361363661361141161333e64e3646463346ee6
9 I-
464e6e5 bebbbebeebbeeoe 6o363440066ee346ee36436eebpbebbboopb beoeobeebe000
666336313e6136313136pleobe3663333663166366beeoeepeoe3616ppleeebeee3316e66
eeaeoo6eabealeoemeooeeoe46eeeebbpoo4646ee66e6leeo646E66eeeab000lbleeeolb
blebpoopeebloblboolftolloolbeeolbb0000lbebbplebpepoombp000elbboo 6133113336
Tiepop666),e3T6beebpobp636433Te364646363364346163e3beeofteepebe66463333e63
0 I-
o6opneleaBebo6pleBeal6pBeoleamBee6TbolloeoBeBBoompe66166366p6pBeemep
Tbpoeomeeeeobeobweoobolebeebeebleblblebbeepleoplebb000bbe000boboebobee
oeo6eoo600366e36631066eooe661361633e6eelp6ee6eeo6e6p6eo6plee6006eo6pool
6ee66e6ee343e6636366343643e3em6434e3e636433463343e3e3e334e36616e664e43e6463e
eobob000lpe4646ee646p33636eopbeollbee6633eoblelleobe66136peeoe33613446e633
ee333e334e36e3e66464e6e366ee6463e634ee6e3446466eee3634e6433e6633661331e3e636
bbebeebebbeebeeebe6p3135666133oebeeebboeboA33366w36161333elebebbooeme
oeeboe600bebeopeopeebbbAmeoboo6T6poeoebellooeeeobeeooeomeoobbeee6666
000bbeebeboeeleboeoffibeebeeoopneeeb000eabobeee6135pboobeamebooblbpabe
ZZZSLO/IZOZdJ/Id tS06LO/ZZOZ OAA
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tccctcggtgaccctctgggaattgaagatagcttggagagccaggactcgatggaattctagctgtgccttctagttg
c
cagccatctgttglitgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaa
atga
ggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggagg
attgggaagacaatagcaggcatgctggggatgcggtgggctctatggtctagaatggcagtggccggtggggaca
gggctgagccagcaccaaccactcagcctttgagatcccgaggctggtctactgctgagaccttttgttagaagagag
gagatcaagcatttgcaaggtttctgagtgtcaaaatatgaatccaagataactctttcacaatcctaacttcatgctg
tct
acaggtccatattttagcctgctttctccatgttcatccgaaaagaaagaaaagctaagggtggtggtcatatttgaaa
tt
agccagatcttaagtttttctgggggaaatttagaagaaaatatggaaaagtgactatgagcaca
In some embodiments, the genome of the invention comprises a nucleotide
sequence that has
at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, at least
98%, or at least 99% identity to SEQ ID NO: 40.
In some embodiments, the genome of the invention comprises the nucleotide
sequence of
SEQ ID NO: 40.
Exemplary nucleotide sequence insert (SEQ ID NO: 40)
gaattcctgacctcttctcttcctcccacaggccgccaccatggccgcctccttcccacctacccttggattgtcctcc
gcc
cctgacgaaattcaacatccccacatcaaattctcggagtggaagttcaagctctttcgcgtgcgctcgttcgaaaaga
cccccgaggaagcccaaaaggagaagaaagactcattcgaaggaaaacccagcctcgaacagtccccggccgt
cctggacaaggccgacgggcagaagcctgtgccgacccagccgctgctgaaagcgcacccgaaattctccaaga
agtttcacgataacgagaaggcccggggaaaggccatccaccaagcaaaccttagacacctgtgccgcatctgtgg
gaactcattcagagccgacgaacataaccggagataccctgtgcatggccctgtcgacggaaagaccctggggctc
ctgagaaagaaggagaagagggcgacatcctggccggacctgatcgcaaaggtgttcagaatcgacgtgaaggc
agatgtggacagcatccacccaaccgagttctgccacaactgctggagcattatgcaccggaagttcagctcagcgc
cctgtgaagtgtacttcccgcgcaacgtgactatggagtggcatccacacactccgtcctgcgacatctgtaacactgc
t
cggcgcggactcaagaggaagtccctgcagccgaatctgcagctgagcaagaagcttaagaccgtgctggaccag
gctcggcaggcccgccagcacaagcgacgcgcccaggcccggatctcatctaaggatgtgatgaagaagatcgc
caattgcagcaaaatccacctgtctaccaagctgctggcggtggacttcccggagcacttcgtgaagtccatcagctgt
cagatctgcgagcatattctcgccgaccccgtggagactaattgcaagcacgtgttctgccgcgtgtgcatcctgcgct
gcctgaaggtcatgggctcctattgcccttcctgccggtacccctgtttccctactgatctggagtccccggtcaagtc
ctt
cttgtccgtgctgaactccctgatggtcaaatgtcccgcaaaggagtgcaatgaggaagtgtccctggaaaagtacaa
ccaccacatcagcagccacaaggagtccaaagaaatctttgtgcacattaacaagggeggtcggccccggcagca
tctgctctcgctgactcgccgggcccagaagcacaggctccgggagctgaagctgcaagtcaaggccttcgccgac
aaggaagagggaggagatgtgaagtccgtgtgcatgaccctgtttttgctggcgctgcgggctcggaacgaacaca
gacaagctgatgaactggaggccatcatgcagggcaaaggatcgggactccagccggctgtgtgtctcgccatccg
cgtcaacacattcctctcatgctcccaataccacaagatgtacaggactgtgaaggccatcaccggacggcagatctt
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tcagccactccacgcccttcggaacgcagaaaaggtcttgctgccgggataccatcatttcgaatggcagccgccctt
gaaaaacgtgtcctcgtccaccgacgtgggcattattgatgggctgagcggcctgtcctcctctgtggatgactaccct
g
tggataccatcgccaaacggttcagatacgattccgcgctggtgtcggccctgatggacatggaggaggacatcctg
gagggaatgagatcacaagatctggacgactacctcaacgggcccttcacggtggtggtcaaggaatcgtgcgatg
gaatgggcgacgtgtcggagaagcacggttccggacctgtggtgccggaaaaggccgtgcgcttctccttcaccatc
atgaagatcaccattgcgcatagctcccagaacgtcaaagtgttcgaagaggccaagccgaactcagagctctgctg
caagccgctgtgcctgatgttggcggacgagagcgatcacgaaaccctgaccgccattctgtcgcctctgatcgcgg
agagggaggccatgaagtcctccgaactgatgctggagctgggcggtattttgcggacttttaagttcatcttccgggg
a
accggttatgacgaaaagctcgtgcgcgaagtggagggcctggaagcctcaggctccgtctacatctgcactctctgc
gacgccacccggctggaggcgtcacagaatcttgtgttccactcgatcactaggtcccacgcggagaacctggaac
gctatgaggtctggcgctctaacccataccacgaatccgtggaagaacttcgggacagagtgaagggagtgtcagc
aaagcctlicattgaaaccgtgcctagcatcgacgccctccattgcgacatcggcaacgccgccgagttctacaagat
cttccagcttgagatcggggaagtgtacaagaacccgaacgcctccaaggaagaaagaaagcggtggcaggcta
cccttgacaaacacctccgcaagaagatgaacctgaagcccattatgcggatgaacggaaacttcgctaggaagct
gatgactaaggaaacggtcgacgcggtctgtgaactgatccccagcgaagaacgacatgaagcgctgcgcgaact
catggacctgtacctgaagatgaagcctgtctggcggagctcgtgccctgccaaggagtgcccggagtcgctgtgtca
gtacagctttaacagccaaaggttcgcagagctgctgtcgaccaagttcaagtacagatacgaaggaaagattacca
actacttccacaagactctcgctcacgtgcccgagattatcgaacgcgatggttccatcggggcctgggcctccgagg
gcaacgagtcgggcaacaagttgttccgccggtttagaaagatgaacgcccgccagtccaagtgctacgaaatgga
agatgtgctgaagcatcactggctgtatacctccaagtacctccagaagttcatgaacgcacataacgccctcaagac
ctccgggttcaccatgaacccccaggcctccctcggtgaccctctgggaattgaagatagcttggagagccaggactc
gatggaattctagctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttecttgaccctggaag
gtgc
cactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggt
ggg
gtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggtc
taga
Variants, derivatives, analogues, and fragments
In addition to the specific proteins and nucleotides mentioned herein, the
invention also
encompasses variants, derivatives, and fragments thereof.
In the context of the invention, a "variant" of any given sequence is a
sequence in which the
specific sequence of residues (whether amino acid or nucleic acid residues)
has been modified
in such a manner that the polypeptide or polynucleotide in question retains at
least one of its
endogenous functions. For example, a variant of RAG1 may retain the ability to
form a RAG
complex, mediate DNA-binding to the RSS, and introduce a double-strand break
between the
RSS and the adjacent coding segment. A variant sequence can be obtained by
addition,
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deletion, substitution, modification, replacement and/or variation of at least
one residue
present in the naturally occurring polypeptide or polynucleotide.
The term "derivative" as used herein in relation to proteins or polypeptides
of the invention
includes any substitution of, variation of, modification of, replacement of,
deletion of and/or
addition of one (or more) amino acid residues from or to the sequence,
providing that the
resultant protein or polypeptide retains at least one of its endogenous
functions. For example,
a derivative of RAG1 may retain the ability to form a RAG complex, mediate DNA-
binding to
the RSS, and introduce a double-strand break between the RSS and the adjacent
coding
segment.
Typically, amino acid substitutions may be made, for example from 1, 2 or 3,
to 10 or 20
substitutions, provided that the modified sequence retains the required
activity or ability. Amino
acid substitutions may include the use of non-naturally occurring analogues.
Proteins used in the invention may also have deletions, insertions or
substitutions of amino
acid residues which produce a silent change and result in a functionally
equivalent protein.
Deliberate amino acid substitutions may be made on the basis of similarity in
polarity, charge,
solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of
the residues as long
as the endogenous function is retained. For example, negatively charged amino
acids include
aspartic acid and glutamic acid; positively charged amino acids include lysine
and arginine;
and amino acids with uncharged polar head groups having similar hydrophilicity
values include
asparagine, glutamine, serine, threonine and tyrosine.
Conservative substitutions may be made, for example according to the table
below. Amino
acids in the same block in the second column and in the same line in the third
column may be
substituted for each other:
ALI PHATIC Non-polar G A P
ILV
Polar - uncharged CSTM
NQ
Polar - charged D E
K R H
AROMATIC F W Y
Typically, a variant may have a certain identity with the wild type amino acid
sequence or the
wild type nucleotide sequence.
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In the present context, a variant sequence is taken to include an amino acid
sequence which
may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, suitably at least
95%, 96% or
97% or 98% or 99% identical to the subject sequence. Although a variant can
also be
considered in terms of similarity (i.e. amino acid residues having similar
chemical
properties/functions), in the context of the present invention it is preferred
to express in terms
of sequence identity.
In the present context, a variant sequence is taken to include a nucleotide
sequence which
may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, suitably at least
95%, 96% or
97% or 98% or 99% identical to the subject sequence. Although a variant can
also be
considered in terms of similarity, in the context of the present invention it
is preferred to
express it in terms of sequence identity.
Suitably, reference to a sequence which has a percent identity to any one of
the SEQ ID NOs
detailed herein refers to a sequence which has the stated percent identity
over the entire
length of the SEQ ID NO referred to.
Sequence identity comparisons can be conducted by eye, or more usually, with
the aid of
readily available sequence comparison programs. These commercially available
computer
programs can calculate percent identity between two or more sequences.
Percent identity may be calculated over contiguous sequences, i.e. one
sequence is aligned
with the other sequence and each amino acid or nucleotide in one sequence is
directly
compared with the corresponding amino acid or nucleotide in the other
sequence, one residue
at a time. This is called an "ungapped" alignment. Typically, such ungapped
alignments are
performed only over a relatively short number of residues.
Although this is a very simple and consistent method, it fails to take into
consideration that, for
example, in an otherwise identical pair of sequences, one insertion or
deletion in the amino
acid or nucleotide sequence may cause the following residues or codons to be
put out of
alignment, thus potentially resulting in a large reduction in percent identity
when a global
alignment is performed. Consequently, most sequence comparison methods are
designed to
produce optimal alignments that take into consideration possible insertions
and deletions
without penalising unduly the overall identity score. This is achieved by
inserting "gaps" in the
sequence alignment to try to maximise local identity.
However, these more complex methods assign "gap penalties" to each gap that
occurs in the
alignment so that, for the same number of identical amino acids or
nucleotides, a sequence
alignment with as few gaps as possible, reflecting higher relatedness between
the two
compared sequences, will achieve a higher score than one with many gaps.
"Affine gap costs"
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are typically used that charge a relatively high cost for the existence of a
gap and a smaller
penalty for each subsequent residue in the gap. This is the most commonly used
gap scoring
system. High gap penalties will of course produce optimised alignments with
fewer gaps. Most
alignment programs allow the gap penalties to be modified. However, it is
preferred to use the
default values when using such software for sequence comparisons. For example
when using
the GCG Wisconsin Bestfit package the default gap penalty for amino acid
sequences is -12
for a gap and -4 for each extension.
Calculation of maximum percent identity therefore firstly requires the
production of an optimal
alignment, taking into consideration gap penalties. A suitable computer
program for carrying
out such an alignment is the GCG Wisconsin Bestfit package (University of
Wisconsin, USA;
Devereux et al. (1984) Nucleic Acids Research 12: 387). Examples of other
software that can
perform sequence comparisons include, but are not limited to, the BLAST
package (see
Ausubel et al. (1999) ibid ¨ Ch. 18), FASTA (Atschul et al. (1990) J. Mol.
Biol. 403-410),
EMBOSS Needle (Madeira, F., et al., 2019. Nucleic acids research, 47(W1),
pp.W636-W641)
and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are
available for
offline and online searching (see Ausubel et al. (1999) ibid, pages 7-58 to 7-
60). However, for
some applications, it is preferred to use the GCG Bestfit program. Another
tool, BLAST 2
Sequences, is also available for comparing protein and nucleotide sequences
(FEMS
Microbiol. Lett. (1999) 174(2):247-50; FEMS Microbiol. Lett. (1999) 177(1):187-
8).
Although the final percent identity can be measured, the alignment process
itself is typically
not based on an all-or-nothing pair comparison. Instead, a scaled similarity
score matrix is
generally used that assigns scores to each pairwise comparison based on
chemical similarity
or evolutionary distance. An example of such a matrix commonly used is the
BLOSUM62
matrix (the default matrix for the BLAST suite of programs). GCG Wisconsin
programs
generally use either the public default values or a custom symbol comparison
table if supplied
(see the user manual for further details). For some applications, it is
preferred to use the public
default values for the GCG package, or in the case of other software, the
default matrix, such
as BLOSUM62.
Once the software has produced an optimal alignment, it is possible to
calculate percent
sequence identity. The software typically does this as part of the sequence
comparison and
generates a numerical result. The percent sequence identity may be calculated
as the number
of identical residues as a percentage of the total residues in the SEQ ID NO
referred to.
"Fragments" are also variants and the term typically refers to a selected
region of the
polypeptide or polynucleotide that is of interest either functionally or, for
example, in an assay.
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"Fragment" thus refers to an amino acid or nucleic acid sequence that is a
portion of a full-
length polypeptide or polynucleotide.
Such variants, derivatives, and fragments may be prepared using standard
recombinant DNA
techniques such as site-directed mutagenesis. Where insertions are to be made,
synthetic
DNA encoding the insertion together with 5' and 3' flanking regions
corresponding to the
naturally-occurring sequence either side of the insertion site may be made.
The flanking
regions will contain convenient restriction sites corresponding to sites in
the naturally-
occurring sequence so that the sequence may be cut with the appropriate
enzyme(s) and the
synthetic DNA ligated into the cut. The DNA is then expressed in accordance
with the invention
to make the encoded protein. These methods are only illustrative of the
numerous standard
techniques known in the art for manipulation of DNA sequences and other known
techniques
may also be used.
Vector
In one aspect, the present invention provides a vector comprising the
polynucleotide of the
invention.
The vector may be suitable for editing a genome using the polynucleotide of
the invention.
The vector may be used to deliver the polynucleotide into the cell.
Subsequently, the
nucleotide sequence insert can be introduced into a genome at a site of a
double strand break
(DSB) by homology-directed repair (H DR).
The vector of the present invention may be capable of transducing mammalian
cells, for
example human cells. Suitably, the vector of the present invention is capable
of transducing
HSCs, HPCs, and/or LPCs. Suitably, the vector of the present invention is
capable of
transducing 0D34+ cells. Suitably, the vector of the present invention is
capable of
transducing NALM6, K562, and/or other human cell lines (e.g. Molt4, U937,
etc.). Suitably, the
vector of the present invention is capable of transducing T cells.
Suitably, the vector of the present invention is a viral vector. The vector of
the invention may
be an adeno-associated viral (AAV) vector, although it is contemplated that
other viral vectors
may be used e.g. lentiviral vectors (e.g. IDLV vectors), or single or double
stranded DNA.
The vector of the present invention may be in the form of a viral vector
particle. Suitably, the
viral vector of the present invention is in the form of an AAV vector
particle. Suitably, the viral
vector of the present invention is in the form of a lentiviral vector
particle, for example an IDLV
vector particle.
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Methods of preparing and modifying viral vectors and viral vector particles,
such as those
derived from AAV, are well known in the art. Suitable methods are described in
Ayuso, E., et
al., 2010. Current gene therapy, 10(6), pp.423-436, Merten, OW., et al., 2016.
Molecular
Therapy-Methods & Clinical Development, 3, p.16017; and Nadeau, I. and Kamen,
A., 2003.
Biotechnology advances, 20(7-8), pp.475-489.
Adeno-associated viral (AAV) vectors
The vector of the present invention may be an adeno-associated viral (AAV)
vector. Optionally,
the vector is an AAV6 vector. The vector of the present invention may be in
the form of an
AAV vector particle. Optionally, the vector is in the form of an AAV6 vector
particle.
The AAV vector or AAV vector particle may comprise an AAV genome or a fragment
or
derivative thereof. An AAV genome is a polynucleotide sequence, which may
encode functions
needed for production of an AAV particle. These functions include those
operating in the
replication and packaging cycle of AAV in a host cell, including encapsidation
of the AAV
genome into an AAV particle. Naturally occurring AAVs are replication-
deficient and rely on
the provision of helper functions in trans for completion of a replication and
packaging cycle.
Accordingly, the AAV genome of the AAV vector of the invention is typically
replication-
deficient.
The AAV genome may be in single-stranded form, either positive or negative-
sense, or
alternatively in double-stranded form. The use of a double-stranded form
allows bypass of the
DNA replication step in the target cell and so can accelerate transgene
expression.
AAVs occurring in nature may be classified according to various biological
systems. The AAV
genome may be from any naturally derived serotype, isolate or clade of AAV.
AAV may be referred to in terms of their serotype. A serotype corresponds to a
variant
subspecies of AAV which, owing to its profile of expression of capsid surface
antigens, has a
distinctive reactivity which can be used to distinguish it from other variant
subspecies.
Typically, an AAV vector particle having a particular AAV serotype does not
efficiently cross-
react with neutralising antibodies specific for any other AAV serotype. AAV
serotypes include
AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11. The AAV
vector of the invention may be an AAV6 serotype.
AAV may also be referred to in terms of Glades or clones. This refers to the
phylogenetic
relationship of naturally derived AAVs, and typically to a phylogenetic group
of AAVs which
can be traced back to a common ancestor, and includes all descendants thereof
Additionally,
AAVs may be referred to in terms of a specific isolate, i.e. a genetic isolate
of a specific AAV
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found in nature. The term genetic isolate describes a population of AAVs which
has undergone
limited genetic mixing with other naturally occurring AAVs, thereby defining a
recognisably
distinct population at a genetic level.
Typically, the AAV genome of a naturally derived serotype, isolate or clade of
AAV comprises
at least one inverted terminal repeat sequence (ITR). An ITR sequence acts in
cis to provide
a functional origin of replication and allows for integration and excision of
the vector from the
genome of a cell. ITRs may be the only sequences required in cis next to the
therapeutic gene.
Suitably, one or more ITR sequences flank the polynucleotide of the invention.
The AAV genome may also comprise packaging genes, such as rep and/or cap genes
which
encode packaging functions for an AAV particle. A promoter may be operably
linked to each
of the packaging genes. Specific examples of such promoters include the p5,
p19 and p40
promoters. For example, the p5 and p19 promoters are generally used to express
the rep
gene, while the p40 promoter is generally used to express the cap gene. The
rep gene
encodes one or more of the proteins Rep78, Rep68, Rep52 and Rep40 or variants
thereof.
The cap gene encodes one or more capsid proteins such as VP1, VP2 and VP3 or
variants
thereof.
The AAV genome may be the full genome of a naturally occurring AAV. For
example, a vector
comprising a full AAV genome may be used to prepare an AAV vector or vector
particle.
Suitably, the AAV genome is derivatised for the purpose of administration to
patients. Such
derivatisation is standard in the art and the invention encompasses the use of
any known
derivative of an AAV genome, and derivatives which could be generated by
applying
techniques known in the art. The AAV genome may be a derivative of any
naturally occurring
AAV. Suitably, the AAV genome is a derivative of AAV1, AAV2, AAV3, AAV4, AAV5,
AAV6,
AAV7, AAV8, AAV9, AAV10, or AAV11. Suitably, the AAV genome is a derivative of
AAV6.
Derivatives of an AAV genome include any truncated or modified forms of an AAV
genome
which allow for expression of a transgene from an AAV vector of the invention
in vivo.
Typically, it is possible to truncate the AAV genome significantly to include
minimal viral
sequence yet retain the above function. This may reduce the risk of
recombination of the
vector with wild-type virus, and avoid triggering a cellular immune response
by the presence
of viral gene proteins in the target cell.
Typically, a derivative will include at least one inverted terminal repeat
sequence (ITR),
optionally more than one ITR, such as two ITRs or more. One or more of the
ITRs may be
derived from AAV genomes having different serotypes, or may be a chimeric or
mutant ITR.
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A suitable mutant ITR is one having a deletion of a trs (terminal resolution
site). This deletion
allows for continued replication of the genome to generate a single-stranded
genome which
contains both coding and complementary sequences, i.e. a self-complementary
AAV genome.
This allows for bypass of DNA replication in the target cell, and so enables
accelerated
transgene expression.
The AAV genome may comprise one or more ITR sequences from any naturally
derived
serotype, isolate or clade of AAV or a variant thereof. The AAV genome may
comprise at least
one, such as two, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,
or
AAV11 ITRs, or variants thereof.
The one or more ITRs may flank the nucleotide sequence of the invention at
either end. The
inclusion of one or more ITRs is can aid concatamer formation of the AAV
vector in the nucleus
of a host cell, for example following the conversion of single-stranded vector
DNA into double-
stranded DNA by the action of host cell DNA polymerases. The formation of such
episomal
concatamers protects the AAV vector during the life of the host cell, thereby
allowing for
prolonged expression of the transgene in vivo.
Suitably, ITR elements will be the only sequences retained from the native AAV
genome in
the derivative. Suitably, a derivative may not include the rep and/or cap
genes of the native
genome and any other sequences of the native genome. This may reduce the
possibility of
integration of the vector into the host cell genome. Additionally, reducing
the size of the AAV
genome allows for increased flexibility in incorporating other sequence
elements (such as
regulatory elements) within the vector in addition to the transgene.
The following portions could therefore be removed in a derivative of the
invention: one inverted
terminal repeat (ITR) sequence, the replication (rep) and capsid (cap) genes.
However,
derivatives may additionally include one or more rep and/or cap genes or other
viral
sequences of an AAV genome. Naturally occurring AAV integrates with a high
frequency at a
specific site on human chromosome 19, and shows a negligible frequency of
random
integration, such that retention of an integrative capacity in the AAV vector
may be tolerated
in a therapeutic setting.
The invention additionally encompasses the provision of sequences of an AAV
genome in
a different order and configuration to that of a native AAV genome. The
invention also
encompasses the replacement of one or more AAV sequences or genes with
sequences
from another virus or with chimeric genes composed of sequences from more than
one
virus. Such chimeric genes may be composed of sequences from two or more
related viral
proteins of different viral species.
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The AAV vector particle may be encapsidated by capsid proteins. Suitably, the
AAV vector
particles may be transcapsidated forms wherein an AAV genome or derivative
having an ITR
of one serotype is packaged in the capsid of a different serotype. The AAV
vector particle also
includes mosaic forms wherein a mixture of unmodified capsid proteins from two
or more
different serotypes makes up the viral capsid. The AAV vector particle also
includes chemically
modified forms bearing ligands adsorbed to the capsid surface. For example,
such ligands
may include antibodies for targeting a particular cell surface receptor.
Where a derivative comprises capsid proteins i.e. VP1, VP2 and/or VP3, the
derivative may
be a chimeric, shuffled or capsid-modified derivative of one or more naturally
occurring AAVs.
In particular, the invention encompasses the provision of capsid protein
sequences from
different serotypes, clades, clones, or isolates of AAV within the same vector
(i.e. a
pseudotyped vector). The AAV vector may be in the form of a pseudotyped AAV
vector
particle.
Chimeric, shuffled or capsid-modified derivatives will be typically selected
to provide one or
more desired functionalities for the AAV vector. Thus, these derivatives may
display increased
efficiency of gene delivery and/or decreased immunogenicity (humoral or
cellular) compared
to an AAV vector comprising a naturally occurring AAV genome. Increased
efficiency of gene
delivery, for example, may be effected by improved receptor or co-receptor
binding at the cell
surface, improved internalisation, improved trafficking within the cell and
into the nucleus,
improved uncoating of the viral particle and improved conversion of a single-
stranded genome
to double-stranded form.
Chimeric capsid proteins include those generated by recombination between two
or more
capsid coding sequences of naturally occurring AAV serotypes. This may be
performed for
example by a marker rescue approach in which non-infectious capsid sequences
of one
serotype are co-transfected with capsid sequences of a different serotype, and
directed
selection is used to select for capsid sequences having desired properties.
The capsid
sequences of the different serotypes can be altered by homologous
recombination within the
cell to produce novel chimeric capsid proteins.
Chimeric capsid proteins also include those generated by engineering of capsid
protein
sequences to transfer specific capsid protein domains, surface loops or
specific amino acid
residues between two or more capsid proteins, for example between two or more
capsid
proteins of different serotypes.
Shuffled or chimeric capsid proteins may also be generated by DNA shuffling or
by error-prone
PCR. Hybrid AAV capsid genes can be created by randomly fragmenting the
sequences of
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related AAV genes e.g. those encoding capsid proteins of multiple different
serotypes and
then subsequently reassembling the fragments in a self-priming polymerase
reaction, which
may also cause crossovers in regions of sequence homology. A library of hybrid
AAV genes
created in this way by shuffling the capsid genes of several serotypes can be
screened to
identify viral clones having a desired functionality. Similarly, error prone
PCR may be used to
randomly mutate AAV capsid genes to create a diverse library of variants which
may then be
selected for a desired property.
The sequences of the capsid genes may also be genetically modified to
introduce specific
deletions, substitutions or insertions with respect to the native wild-type
sequence. In
particular, capsid genes may be modified by the insertion of a sequence of an
unrelated
protein or peptide within an open reading frame of a capsid coding sequence,
or at the N-
and/or C-terminus of a capsid coding sequence. The unrelated protein or
peptide may
advantageously be one which acts as a ligand for a particular cell type,
thereby conferring
improved binding to a target cell or improving the specificity of targeting of
the vector to a
particular cell population. The unrelated protein may also be one which
assists purification of
the viral particle as part of the production process, i.e. an epitope or
affinity tag. The site of
insertion will typically be selected so as not to interfere with other
functions of the viral particle
e.g. internalisation, trafficking of the viral particle.
The capsid protein may be an artificial or mutant capsid protein. The term
"artificial capsid" as
used herein means that the capsid particle comprises an amino acid sequence
which does
not occur in nature or which comprises an amino acid sequence which has been
engineered
(e.g. modified) from a naturally occurring capsid amino acid sequence. In
other words the
artificial capsid protein comprises a mutation or a variation in the amino
acid sequence
compared to the sequence of the parent capsid from which it is derived where
the artificial
capsid amino acid sequence and the parent capsid amino acid sequences are
aligned. The
AAV vector particle may comprise an AAV6 capsid protein.
Retroviral and lentiviral vectors
The vector of the present invention may be a retroviral vector or a lentiviral
vector. The vector
of the present invention may be a retroviral vector particle or a lentiviral
vector particle_
A retroviral vector may be derived from or may be derivable from any suitable
retrovirus. A
large number of different retroviruses have been identified. Examples include
murine
leukaemia virus (MLV), human T-cell leukaemia virus (HTLV), mouse mammary
tumour virus
(MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney
murine
leukaemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney
murine
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sarcoma virus (Mo-MSV), Abelson murine leukaemia virus (A-MLV), avian
myelocytomatosis
virus-29 (MC29) and avian erythroblastosis virus (AEV).
Retroviruses may be broadly divided into two categories, "simple" and
"complex". Retroviruses
may be even further divided into seven groups. Five of these groups represent
retroviruses
with oncogenic potential. The remaining two groups are the lentiviruses and
the spumaviruses.
The basic structure of retrovirus and lentivirus genomes share many common
features such
as a 5' LTR and a 3' LTR. Between or within these are located a packaging
signal to enable
the genome to be packaged, a primer binding site, integration sites to enable
integration into
a host cell genome, and gag, pol and env genes encoding the packaging
components ¨ these
are polypeptides required for the assembly of viral particles. Lentiviruses
have additional
features, such as rev and RRE sequences in HIV, which enable the efficient
export of RNA
transcripts of the integrated provirus from the nucleus to the cytoplasm of an
infected target
cell.
In the provirus, these genes are flanked at both ends by regions called long
terminal repeats
(LTRs). The LTRs are responsible for proviral integration and transcription.
LTRs also serve
as enhancer-promoter sequences and can control the expression of the viral
genes.
The LTRs themselves are identical sequences that can be divided into three
elements: U3, R
and U5. U3 is derived from the sequence unique to the 3' end of the RNA. R is
derived from
a sequence repeated at both ends of the RNA. U5 is derived from the sequence
unique to the
5' end of the RNA. The sizes of the three elements can vary considerably among
different
retrovi ruses.
In a defective retroviral vector genome gag, pol and env may be absent or not
functional.
In a typical retroviral vector, at least part of one or more protein coding
regions essential for
replication may be removed from the virus. This makes the viral vector
replication-defective.
Portions of the viral genome may also be replaced by a library encoding
candidate modulating
moieties operably linked to a regulatory control region and a reporter moiety
in the vector
genome in order to generate a vector comprising candidate modulating moieties
which is
capable of transducing a target host cell and/or integrating its genome into a
host genome.
Lentivirus vectors are part of the larger group of retroviral vectors. In
brief, lentiviruses can be
divided into primate and non-primate groups. Examples of primate lentiviruses
include but are
not limited to human immunodeficiency virus (HIV), the causative agent of
human acquired
immunodeficiency syndrome (AIDS); and simian immunodeficiency virus (SIV)_
Examples of
non-primate lentiviruses include the prototype "slow virus" visna/maedi virus
(VMV), as well
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as the related caprine arthritis-encephalitis virus (CAEV), equine infectious
anaemia virus
(EIAV), and the more recently described feline immunodeficiency virus (FIV)
and bovine
immunodeficiency virus (BIV).
The lentivirus family differs from retroviruses in that lentiviruses have the
capability to infect
both dividing and non-dividing cells. In contrast, other retroviruses, such as
MLV, are unable
to infect non-dividing or slowly dividing cells such as those that make up,
for example, muscle,
brain, lung and liver tissue.
A lentiviral vector, as used herein, is a vector which comprises at least one
component part
derivable from a lentivirus. Suitably, that component part is involved in the
biological
mechanisms by which the vector infects cells, expresses genes or is
replicated.
The lentiviral vector may be a "primate" vector. The lentiviral vector may be
a "non-primate"
vector (i.e. derived from a virus which does not primarily infect primates,
especially humans).
Examples of non-primate lentiviruses may be any member of the family of
lentiviridae which
does not naturally infect a primate.
As examples of lentivirus-based vectors, HIV-1- and HIV-2-based vectors are
described
below.
The HIV-1 vector contains cis-acting elements that are also found in simple
retroviruses. It has
been shown that sequences that extend into the gag open reading frame are
important for
packaging of HIV-1. Therefore, HIV-1 vectors often contain the relevant
portion of gag in which
the translational initiation codon has been mutated. In addition, most HIV-1
vectors also
contain a portion of the env gene that includes the RRE. Rev binds to RRE,
which permits the
transport of full-length or singly spliced mRNAs from the nucleus to the
cytoplasm. In the
absence of Rev and/or RRE, full-length HIV-1 RNAs accumulate in the nucleus.
Alternatively,
a constitutive transport element from certain simple retroviruses such as
Mason-Pfizer
monkey virus can be used to relieve the requirement for Rev and RRE. Efficient
transcription
from the HIV-1 LTR promoter requires the viral protein Tat.
Most HIV-2-based vectors are structurally very similar to HIV-1 vectors.
Similar to HIV-1-based
vectors, HIV-2 vectors also require RRE for efficient transport of the full-
length or singly spliced
viral RNAs.
Optionally, the viral vector used in the present invention has a minimal viral
genome.
By "minimal viral genome" it is to be understood that the viral vector has
been manipulated so
as to remove the non-essential elements and to retain the essential elements
in order to
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provide the required functionality to infect, transduce and deliver a
nucleotide sequence of
interest to a target host cell. Further details of this strategy can be found
in WO 1998/017815.
Optionally, the plasmid vector used to produce the viral genome within a host
cell/packaging
cell will have sufficient lentiviral genetic information to allow packaging of
an RNA genome, in
the presence of packaging components, into a viral particle which is capable
of infecting a
target cell, but is incapable of independent replication to produce infectious
viral particles
within the final target cell. Optionally, the vector lacks a functional gag-
pol and/or env gene
and/or other genes essential for replication.
However, the plasmid vector used to produce the viral genome within a host
cell/packaging
cell will also include transcriptional regulatory control sequences operably
linked to the
lentiviral genome to direct transcription of the genome in a host
cell/packaging cell. These
regulatory sequences may be the natural sequences associated with the
transcribed viral
sequence (i.e. the 5' U3 region), or they may be a heterologous promoter, such
as another
viral promoter (e.g. the CMV promoter).
The vectors may be self-inactivating (SIN) vectors in which the viral enhancer
and promoter
sequences have been deleted. SIN vectors can be generated and transduce non-
dividing cells
in vivo with an efficacy similar to that of wild-type vectors. The
transcriptional inactivation of
the long terminal repeat (LTR) in the SIN provirus should prevent mobilisation
by replication-
competent virus. This should also enable the regulated expression of genes
from internal
promoters by eliminating any cis-acting effects of the LTR.
The vectors may be integration-defective. Integration defective lentiviral
vectors (IDLVs) can
be produced, for example, either by packaging the vector with catalytically
inactive integrase
(such as an HIV integrase bearing the D64V mutation in the catalytic site) or
by modifying or
deleting essential att sequences from the vector LTR, or by a combination of
the above.
Adenoviral vectors
The vector of the present invention may be an adenoviral vector. The vector of
the present
invention may be an adenoviral vector particle.
The adenovirus is a double-stranded, linear DNA virus that does not go through
an RNA
intermediate. There are over 50 different human serotypes of adenovirus
divided into 6
subgroups based on the genetic sequence homology. The natural targets of
adenovirus are
the respiratory and gastrointestinal epithelia, generally giving rise to only
mild symptoms.
Serotypes 2 and 5 (with 95% sequence homology) are most commonly used in
adenoviral
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vector systems and are normally associated with upper respiratory tract
infections in the
young.
Adenoviruses have been used as vectors for gene therapy and for expression of
heterologous
genes. The large (36 kb) genome can accommodate up to 8 kb of foreign insert
DNA and is
able to replicate efficiently in complementing cell lines to produce very high
titres of up to 1012.
Adenovirus is thus one of the best systems to study the expression of genes in
primary non-
replicative cells.
The expression of viral or foreign genes from the adenovirus genome does not
require a
replicating cell. Adenoviral vectors enter cells by receptor mediated
endocytosis. Once inside
the cell, adenovirus vectors rarely integrate into the host chromosome.
Instead, they function
episomally (independently from the host genome) as a linear genome in the host
nucleus.
Hence the use of recombinant adenovirus alleviates the problems associated
with random
integration into the host genome.
Herpes simplex viral vector
The vector of the present invention may be a herpes simplex viral vector. The
vector of the
present invention may be a herpes simplex viral vector particle.
Herpes simplex virus (HSV) is a neurotropic DNA virus with favorable
properties as a gene
delivery vector. HSV is highly infectious, so HSV vectors are efficient
vehicles for the delivery
of exogenous genetic material to cells. Viral replication is readily disrupted
by null mutations
in immediate early genes that in vitro can be complemented in trans, enabling
straightforward
production of high-titre pure preparations of non-pathogenic vector. The
genome is large (152
Kb) and many of the viral genes are dispensable for replication in vitro,
allowing their
replacement with large or multiple transgenes. Latent infection with wild-type
virus results in
episomal viral persistence in sensory neuronal nuclei for the duration of the
host lifetime. The
vectors are non-pathogenic, unable to reactivate and persist long-term. The
latency active
promoter complex can be exploited in vector design to achieve long-term stable
transgene
expression in the nervous system. HSV vectors transduce a broad range of
tissues because
of the wide expression pattern of the cellular receptors recognized by the
virus. Increasing
understanding of the processes involved in cellular entry has allowed
targeting the tropism of
HSV vectors.
Vaccinia virus vectors
The vector of the present invention may be a vaccinia viral vector. The vector
of the present
invention may be a vaccinia viral vector particle.
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Vaccinia virus is large enveloped virus that has an approximately 190 kb
linear, double-
stranded DNA genome. Vaccinia virus can accommodate up to approximately 25 kb
of foreign
DNA, which also makes it useful for the delivery of large genes.
A number of attenuated vaccinia virus strains are known in the art that are
suitable for gene
therapy applications, for example the MVA and NYVAC strains.
RNA-guided gene editing
The vector of the present invention may be used to deliver a polynucleotide
into a cell.
Subsequently, a nucleotide sequence insert can be introduced into the cell's
genome at a site
of a double strand break (DSB) by homology-directed repair (HDR). The site of
the double-
strand break (DSB) can be introduced specifically by any suitable technique,
for example by
using an RNA-guided gene editing system.
An "RNA-guided gene editing system" can be used to introduce a DSB and
typically comprises
a guide RNA and a RNA-guided nuclease. A CRISPR/Cas9 system is an example of a
commonly used RNA-guided gene editing system, but other RNA-guided gene
editing
systems may also be used.
Guide RNAs
A "guide RNA" (gRNA) confers target sequence specificity to a RNA-guided
nuclease. Guide
RNAs are non-coding short RNA sequences which bind to the complementary target
DNA
sequences. For example, in the CRISPR/Cas9 system, guide RNA first binds to
the Cas9
enzyme and the gRNA sequence guides the resulting complex via base-pairing to
a specific
location on the DNA, where Cas9 performs its nuclease activity by cutting the
target DNA
strand.
The term "guide RNA" encompasses any suitable gRNA that can be used with any
RNA-
guided nuclease, and not only those gRNAs that are compatible with a
particular nuclease
such as Cas9.
The guide RNA may comprise a trans-activating CRISPR RNA (tracrRNA) that
provides the
stem loop structure and a target-specific CRISPR RNA (crRNA) designed to
cleave the gene
target site of interest. The tracrRNA and crRNA may be annealed, for example
by heating
them at 95 C for 5 minutes and letting them slowly cool down to room
temperature for 10
minutes. Alternatively, the guide RNA may be a single guide RNA (sgRNA) that
consists of
both the crRNA and tracrRNA as a single construct.
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The guide RNA may comprise of a 3'-end, which forms a scaffold for nuclease
binding, and a
5'-end which is programmable to target different DNA sites. For example, the
targeting
specificity of CRISPR-Cas9 may be determined by the 15-25 bp sequence at the 5
end of the
guide RNA. The desired target sequence typically precedes a protospacer
adjacent motif
(PAM) which is a short DNA sequence usually 2-6 bp in length that follows the
DNA region
targeted for cleavage by the CRISPR system, such as CRISPR-Cas9. The PAM is
required
for a Cas nuclease to cut and is typically found 3-4 bp downstream from the
cut site. After
base pairing of the guide RNA to the target, Cas9 mediates a double strand
break about 3-nt
upstream of PAM.
Numerous tools exist for designing guide RNAs (e.g. Cui, Y., et al., 2018.
Interdisciplinary
Sciences: Computational Life Sciences, 10(2), pp.455-465). For example, COSMID
is a web-
based tool for identifying and validating guide RNAs (Cradick TJ, et al. Mol
Ther - Nucleic
Acids. 2014;3(12):e214).
A list of exemplary guide RNAs for use in the present invention is provided
below in Table 4.
Table 4 - Exemplary guide RNAs
Guide Sequence +1- DSB
site
strand
9 TCAGATGGCAATGTCGAGA (SEQ ID NO: 41) +
chr 11: 36569296-36569297
1 TTTTCCGGATCGATGTGA (SEQ ID NO: 42) +
chr 11: 36573791-36573792
2 GACATCTCTGCCGCATCTG (SEQ ID NO: 43) +
chr 11: 36573642-36573643
3 GTGGGTGCTGAATTTCATC (SEQ ID NO: 44) -
chr 11: 36573352-36573353
4 GATTGTGGGCCAAGTAACG (SEQ ID NO: 45) +
chr 11: 36569081-36569082
5 GAAAGTCACTGTTGGTCGA (SEQ ID NO: 46) -
chr 11: 36572473-36572474
6 CAATTTTGAGGTGTTCGTT (SEQ ID NO: 47) +
chr 11: 36571459-36571460
7 GGGTTGAGTTCAACCTAAG (SEQ ID NO: 48) +
chr 11: 36571367-36571368
8 TTAGCCTCATTGTACTAGC (SEQ ID NO: 49) -
chr 11: 36572860-36572861
10 GCAATTTTGAGGTGTTCGT (SEQ ID NO: 50) +
chr 11: 36571458-36571459
11 ACCAGCCTCGGGATCTCAA (SEQ ID NO: 51) -
chr 11: 36569352-36569353
12 TCAAATCAGTCGGGTTTCC (SEQ ID NO: 52) +
chr 11: 36572376-36572377
In one aspect, the present invention provides a guide RNA comprising or
consisting of a
nucleotide sequence that has at least 90% identity or at least 95% identity to
any of SEQ ID
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NOs: 41-52, optionally wherein the guide RNA comprises or consists of a
nucleotide sequence
that has at least 90% identity or at least 95% identity to SEQ ID NO: 41.
In some embodiments, the guide RNA comprises or consists of the nucleotide
sequence of
any of SEQ ID NOs: 41-52, optionally wherein the guide RNA comprises or
consists of the
nucleotide sequence of SEQ ID NO: 41.
For example, sequences for guides 9, 3 and 7 may be extended as shown below,
for example
when used as crRNA:
Guide Sequence +1- DSB
site
strand
9 GTCAGATGGCAATGTCGAGA (SEQ ID NO:
chr 11: 36569296-36569297
53)
3 TGTGGGTGCTGAATTTCATC (SEQ ID NO: 54) -
chr 11: 36573352-36573353
7 GGGGTTGAGTTCAACCTAAG (SEQ ID NO:
chr 11: 36571367-36571368
55)
In one aspect, the present invention provides a guide RNA comprising or
consisting of a
nucleotide sequence that has at least 90% identity or at least 95% identity to
any of SEQ ID
NOs: 53-55, optionally wherein the guide RNA comprises or consists of a
nucleotide sequence
that has at least 90% identity or at least 95% identity to SEQ ID NO: 53.
In some embodiments, the guide RNA comprises or consists of the nucleotide
sequence of
any of SEQ ID NOs: 53-55, optionally wherein the guide RNA comprises or
consists of the
nucleotide sequence of SEQ ID NO: 53.
Suitably, the guide RNA is chemically modified. The chemical modification may
enhance the
stability of the guide RNA. For example, from one to five (e.g. three) of the
terminal nucleotides
at 5' end and/or 3' end of the guide RNA may be chemically modified to enhance
stability.
Any chemical modification which enhances the stability of the guide RNA may be
used. For
example, the chemical modification may be modification with 2'-0-methyl 3'-
phosphorothioate,
as described in Hendel A, et al. Nat Biotechnol. 2015;33(9):985-9.
RNA-guided nuclease
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A "nuclease" is an enzyme that can cleave the phosphodiester bond present
within a
polynucleotide chain. Suitably, the nuclease is an endonuclease. Endonucleases
are capable
of breaking the bond from the middle of a chain.
An "RNA-guided nuclease" is a nuclease which can be directed to a specific
site by a guide
RNA. The present invention can be implemented using any suitable RNA-guided
nuclease,
for example any RNA-guided nuclease described in Murugan, K., et al., 2017.
Molecular cell,
68(1), pp.15-25. RNA-guided nucleases include, but are not limited to, Type ll
CRISPR
nucleases such as Cas9, and Type V CRISPR nucleases such as Cas12a and Cas12b,
as
well as other nucleases derived therefrom. RNA-guided nucleases can be
defined, in broad
terms, by their PAM specificity and cleavage activity.
Suitably, the RNA-guided nuclease is a Type ll CRISPR nuclease, for example a
Cas9
nuclease. Cas9 is a dual RNA-guided endonuclease enzyme associated with the
Clustered
Regularly Interspaced Short Palindromic Repeats (CRISPR) adaptive immune
system. Cas9
nucleases include the well-characterized ortholog from Streptococcus pyogenes
(SpCas9).
SpCas9 and other orthologs (including SaCas9, FnCa9, and AnaCas9) have been
reviewed
by Jiang, F. and Doudna, J.A., 2017. Annual review of biophysics, 46, pp.505-
529.
The RNA-guided nuclease may be in a complex with the guide RNA, i.e. the guide
RNA and
the RNA-guided nuclease may together form a ribonucleoprotein (RNP). Suitably,
the RNP is
a Cas9 RNP. A RNP may be formed by any method known in the art, for example by
incubating
a RNA-guided nuclease with a guide RNA for 5-30 minutes at room temperature.
Delivering
Cas9 as a preassembled RNP can protect the guide RNA from intracellular
degradation thus
improving stability and activity of the RNA-guided nuclease (Kim S, et al.
Genome Res.
2014;24(6)1012-9).
Kit, cornposition, gene-editing system
In one aspect, the present invention provides a kit, composition, or gene-
editing system
comprising the polynucleotide of the invention, the vector of the invention,
and/or the guide
RNA of the invention.
As used herein, a "gene-editing system" is a system which comprises all
components
necessary to edit a genome using the polynucleotide of the invention.
In some embodiments, the kit, composition, or gene-editing system comprises a
polynucleotide and/or vector of the invention and a guide RNA. The guide RNA
may
correspond to the same DSB site targeted by the homology arms. For example, in
some
embodiments the kit, composition, or gene-editing system comprises:
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(i) a polynucleotide comprising from 5' to 3': a first homology region, a
splice acceptor
sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second
homology region, wherein the first homology region is homologous to a region
upstream of chr 11: 36569295 and the second homology region is homologous to a
region downstream of chr 11: 36569298, and/or a vector comprising said
polynucleotide; and a guide RNA which comprises or consists of a nucleotide
sequence that has at least 90% identity, at least 95% identity or 100%
identity to SEQ
ID NO: 41 or 53 (preferably SEQ ID NO: 41);
(ii) a polynucleotide comprising from 5' to 3': a first homology region, a
splice acceptor
sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second
homology region, wherein the first homology region is homologous to a region
upstream of chr 11: 36573790 and the second homology region is homologous to a
region downstream of chr 11: 36573793 and/or a vector comprising said
polynucleotide; and a guide RNA which comprises or consists of a nucleotide
sequence that has at least 90% identity, at least 95% identity or 100%
identity to SEQ
ID NO: 42;
(iii) a polynucleotide comprising from 5' to 3': a first homology region, a
splice acceptor
sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second
homology region, wherein the first homology region is homologous to a region
upstream of chr 11: 36573641 and the second homology region is homologous to a
region downstream of chr 11: 36573644 and/or a vector comprising said
polynucleotide; and a guide RNA which comprises or consists of a nucleotide
sequence that has at least 90% identity, at least 95% identity or 100%
identity to SEQ
ID NO: 43;
(iv) a polynucleotide comprising from 5' to 3': a first homology region, a
splice acceptor
sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second
homology region, wherein the first homology region is homologous to a region
upstream of chr 11: 36573351 and the second homology region is homologous to a
region downstream of chr 11: 36573354 and/or a vector comprising said
polynucleotide; and a guide RNA which comprises or consists of a nucleotide
sequence that has at least 90% identity, at least 95% identity or 100%
identity to SEQ
ID NO: 44 or 54 (preferably SEQ ID NO: 44);
(v) a polynucleotide comprising from 5' to 3': a first homology region, a
splice acceptor
sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second
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homology region, wherein the first homology region is homologous to a region
upstream of chr 11: 36569080 and the second homology region is homologous to a
region downstream of chr 11: 36569083 and/or a vector comprising said
polynucleotide; and a guide RNA which comprises or consists of a nucleotide
sequence that has at least 90% identity, at least 95% identity or 100%
identity to SEQ
ID NO: 45;
(vi) a polynucleotide comprising from 5' to 3': a first homology region, a
splice acceptor
sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second
homology region, wherein the first homology region is homologous to a region
upstream of chr 11: 36572472 and the second homology region is homologous to a
region downstream of chr 11: 36572475 and/or a vector comprising said
polynucleotide; and a guide RNA which comprises or consists of a nucleotide
sequence that has at least 90% identity, at least 95% identity or 100%
identity to SEQ
ID NO: 46;
(vii) a polynucleotide comprising from 5' to 3': a first homology region, a
splice acceptor
sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second
homology region, wherein the first homology region is homologous to a region
upstream of chr 11: 36571458 and the second homology region is homologous to a
region downstream of chr 11: 36571461 and/or a vector comprising said
polynucleotide; and a guide RNA which comprises or consists of a nucleotide
sequence that has at least 90% identity, at least 95% identity or 100%
identity to SEQ
ID NO: 47;
(viii) a polynucleotide comprising from 5' to 3': a first homology region, a
splice acceptor
sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second
homology region, wherein the first homology region is homologous to a region
upstream of chr 11: 36571366 and the second homology region is homologous to a
region downstream of chr 11: 36571369 and/or a vector comprising said
polynucleotide; and a guide RNA which comprises or consists of a nucleotide
sequence that has at least 90% identity, at least 95% identity or 100%
identity to SEQ
ID NO: 48 or 55 (preferably SEQ ID NO: 48);
(ix) a polynucleotide comprising from 5' to 3': a first homology region, a
splice acceptor
sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second
homology region, wherein the first homology region is homologous to a region
upstream of chr 11: 36572859 and the second homology region is homologous to a
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region downstream of chr 11: 36572862 and/or a vector comprising said
polynucleotide; and a guide RNA which comprises or consists of a nucleotide
sequence that has at least 90% identity, at least 95% identity or 100%
identity to SEQ
ID NO: 49;
(x) a polynucleotide comprising from 5' to 3': a first homology region, a
splice acceptor
sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second
homology region, wherein the first homology region is homologous to a region
upstream of chr 11: 36571457 and the second homology region is homologous to a
region downstream of chr 11: 36571460 and/or a vector comprising said
polynucleotide; and a guide RNA which comprises or consists of a nucleotide
sequence that has at least 90% identity, at least 95% identity or 100%
identity to SEQ
ID NO: 50;
(xi) a polynucleotide comprising from 5' to 3': a first homology region, a
splice acceptor
sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second
homology region, wherein the first homology region is homologous to a region
upstream of chr 11: 36569351 and the second homology region is homologous to a
region downstream of chr 11: 36569354 and/or a vector comprising said
polynucleotide; and a guide RNA which comprises or consists of a nucleotide
sequence that has at least 90% identity, at least 95% identity or 100%
identity to SEQ
ID NO: 51; or
(xii) a polynucleotide comprising from 5' to 3': a first homology region, a
splice acceptor
sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second
homology region, wherein the first homology region is homologous to a region
upstream of chr 11: 36572375 and the second homology region is homologous to a
region downstream of chr 11: 36572378 and/or a vector comprising said
polynucleotide; and a guide RNA which comprises or consists of a nucleotide
sequence that has at least 90% identity, at least 95% identity or 100%
identity to SEQ
ID NO: 52.
In some embodiments, the kit, composition, or gene-editing system comprises:
(i) a polynucleotide comprising from 5' to 3': a first homology region, a
splice acceptor
sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second
homology region, wherein the first homology region is homologous to a region
upstream of chr 11: 36569295 and the second homology region is homologous to a
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region downstream of chr 11: 36569298, and/or a vector comprising said
polynucleotide; and
(ii) a guide RNA which comprises or consists of a nucleotide sequence that has
at least
90% identity, at least 95% identity or 100% identity to SEQ ID NO: 41 or 53
(preferably
SEQ ID NO: 41).
In some embodiments, the kit, composition, or gene-editing system comprises:
(i) a polynucleotide comprising from 5' to 3': a first homology region, a
splice acceptor
sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second
homology region, wherein the first homology region is homologous to a region
comprising chr 11: 36569245-36569294 and the second homology region is
homologous to a region comprising chr 11: 36569299-36569348, and/or a vector
comprising said polynucleotide; and
(ii) a guide RNA which comprises or consists of a nucleotide sequence that has
at least
90% identity, at least 95% identity or 100% identity to SEQ ID NO: 41 or 53
(preferably
SEQ ID NO: 41).
In some embodiments, the kit, composition, or gene-editing system comprises:
(i) a polynucleotide comprising from 5' to 3': a first homology region, a
splice acceptor
sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second
homology region, wherein the first homology region comprises or consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90%
identity, at least 95% identity, at least 98% identity, or 100% identity to
SEQ ID NO: 7
and the second homology region comprises or consists of a nucleotide sequence
that
has at least 70% identity, at least 80% identity, at least 90% identity, at
least 95%
identity, at least 98% identity, or 100% identity to SEQ ID NO: 19, and/or a
vector
comprising said polynucleotide; and
(ii) a guide RNA which comprises or consists of a nucleotide sequence that has
at least
90% identity, at least 95% identity or 100% identity to SEQ ID NO: 41 or 53
(preferably
SEQ ID NO: 41).
In some embodiments, the kit, composition, or gene-editing system comprises:
(i) a polynucleotide comprising from 5' to 3': a first homology region, a
splice acceptor
sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second
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homology region, wherein the first homology region comprises or consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90%
identity, at least 95% identity, at least 98% identity, or 100% identity to
SEQ ID NO:
31, or a fragment thereof; and the second homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90%
identity, at least 95% identity, at least 98% identity, or 100% identity to
SEQ ID NO:
32, or a fragment thereof, and/or a vector comprising said polynucleotide; and
(ii) a guide RNA which comprises or consists of a nucleotide sequence that has
at least
90% identity, at least 95% identity or 100% identity to SEQ ID NO: 41 or 53
(preferably
SEQ ID NO: 41).
The kit, composition, or gene-editing system may further comprise an RNA-
guided nuclease.
Suitably, the RNA-guided nuclease corresponds to the guide RNA used. For
example, if the
guide RNA comprises or consists of a nucleotide sequence that has at least 90%
identity, at
least 95% identity or 100% identity to any one of SEQ ID NOs: 41-52, the RNA-
guided
nuclease is suitably a Cas9 endonuclease. For example, if the guide RNA
comprises or
consists of a nucleotide sequence that has at least 90% identity, at least 95%
identity or 100%
identity to any one of SEQ ID NOs: 53-55, the RNA-guided nuclease is suitably
a Cas9
endonuclease.
The RNA-guided nuclease may be in a complex with the guide RNA, i.e. the guide
RNA and
the RNA-guided nuclease together form a ribonucleoprotein (RNP).
Cell
In one aspect, the present invention provides a cell which has been edited
using the
polynucleotide, vector, kit, composition, or gene-editing system of the
present invention.
In a related aspect, the present invention provides a cell comprising the
polynucleotide, vector
and/or genome of the present invention.
Suitably, the cell is an isolated cell. Suitably, the cell is a mammalian
cell, for example a human
cell.
Suitably, the cell is a hematopoietic stem cell (HSC), a hematopoietic
progenitor cell (H PC),
or a lymphoid progenitor cell (LPC). In some embodiments, the cell is a HSC or
a HPC,
optionally the cell is a HSC.
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As used herein "hematopoietic stem cells" are stem cells that have no
differentiation potential
to cells other than hematopoietic cells, "hematopoietic progenitor cells" are
progenitor cells
that have no differentiation potential to cells other than hematopoietic
cells, and "lymphoid
progenitor cells" are progenitor cells that have no differentiation potential
to cells other than
lymphocytes.
The cell can be obtained from any source. The cell may be autologous or
allogeneic. The cell
may be obtained or obtainable from any biological sample, such as peripheral
blood or cord
blood. Peripheral blood may be treated with mobilising agent, i.e. may be
mobilised peripheral
blood. The cell may be a universal cell.
The cell may be isolated or isolatable using commercially available antibodies
that bind to cell
surface antigens, e.g. CD34, using methods known to those of skill in the art.
For example,
the antibodies may be conjugated to magnetic beads and immunological
procedures utilized
to recover the desired cell type. Suitably, the cell is identified by the
presence or absence of
one or more antigenic markers. Suitable antigenic markers include CD34, CD133,
CD90,
CD45, CD4, CD19, CD13, CD3, CD56, CD14, CD61/41, CD135, CD45RA, CD33, CD66b,
CD38, CD45, CD10, CD11c, CD19, CD7, and CD71.
Suitably, the cell is identified by the presence of the antigenic marker 0D34
(CD34+), i.e. the
cell is a CD34+ cell. For example, the cell may be a cord blood CD34+ cell or
a (mobilised)
peripheral blood CD34+ cell. The cell may be a CD34+ HSC, a CD34+ HPC, or a
CD34+ LPC,
optionally the cell is a C034+ HSC.
In some embodiments, the cell is identified by the presence of CD34 and the
presence or
absence or one or more further antigenic markers. The further antigenic
markers may be
selected from one or more of CD133, CD90, CD3, CD56, CD14, CD61/41, CD135,
CD45RA,
CD33, CD66b, CD38, CD45, CD10, CD11c, CD19, CD7, and CD71. For example, the
cell
may be a CD34+CD133+CD90+ cell, a CD34+CD133+CD90- cell, or a CD34+CD133-CD90-
cell.
Suitably, the cell is a NALM6 cell, a K562 cell, or other human cell (e.g. a
Molt4 cell, a U937
cell, etc.). Suitably, the cell is a T cell.
Population of cells
In one aspect, the present invention provides a population or cells comprising
the cell of the
present invention. Suitably, at least 1%, at least 2%, at least 5%, at least
10%, or at least 20%
of the cells in the population of cells are cells of the present invention.
Suitably, the population
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of cells comprises at least 10x105, at least 50x105, or at least 100x105 cells
of the present
invention.
In a related aspect, the present invention provides a population of cells
which have been edited
using the polynucleotide, vector, kit, composition, or gene-editing system of
the present
invention. Suitably, at least 1%, at least 2%, at least 5%, at least 10%, or
at least 20% of the
cells in the population of cells are cells which have been edited using the
polynucleotide,
vector, kit, composition, or gene-editing system of the present invention.
Suitably, the
population of cells comprises at least 10x105, at least 50x105, or at least
100x105 cells which
have been edited using the polynucleotide, vector, kit, composition, or gene-
editing system of
the present invention.
In a related aspect, the present invention provides a population of cells
comprising the
polynucleotide, vector and/or genome of the present invention. Suitably, at
least 1%, at least
2%, at least 5%, at least 10%, or at least 20% of the cells in the population
of cells are cells
comprising the polynucleotide, vector and/or genome of the present invention.
Suitably, the
population of cells comprises at least 10x105, at least 50x105, or at least
100x105 cells
comprising the polynucleotide, vector and/or genome of the present invention.
Suitably, the population of cells are mammalian cells, for example human
cells. The population
of cells may be autologous or allogeneic. Suitably, the population of cells
are obtained or
obtainable from (mobilised) peripheral blood or cord blood. The population of
cells may be
universal cells.
Suitably, at least 50%, at least 60%, at least 70%, or at least 80% of the
population of cells
are HSCs, HPCs, and/or LPCs. Suitably, at least 50%, at least 60%, at least
70%, or at least
80% of the population of cells are CD34+ cells.
In some embodiments, at least 1%, at least 2%, at least 5%, at least 10%, or
at least 20% of
the population of cells are 0D34+ cells comprising the polynucleotide, vector
and/or genome
of the present invention. For example, in some embodiments at least 20% of the
population of
cells are CD34+ cells comprising the genome of the present invention.
In some embodiments, the population of cells comprises at least 10x105, at
least 50x105, or
at least 100x105 CD34+ cells comprising the polynucleotide, vector and/or
genome of the
present invention. For example, in some embodiments the population of cells
comprises at
least 100x105 CD34+ cells comprising the genome of the present invention.
Method of gene editing
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In one aspect, the present invention provides a method of gene editing a cell
or a population
of cells using polynucleotides, vectors, guide RNAs, kits, compositions and/or
gene-editing
system of the present invention. The present invention also provide a
population of gene-
edited cells obtained or obtainable by said methods.
In another aspect the present invention provides use of a polynucleotide,
vector, guide RNA,
kit, composition, and/or gene-editing system of the present invention for gene
editing a cell or
a population of cells.
Suitably, the method of gene editing a cell or a population of cells
comprises:
(a) providing a cell or a population of cells; and
(b) using a kit, composition, and/or gene-editing system described herein to
obtain a
gene-edited cell or a population of gene-edited cells.
For example, the method of gene editing a cell or a population of cells
comprises:
(a) providing a cell or a population of cells; and
(b) delivering an RNA-guided nuclease, a guide RNA, and/or a polynucleotide or
vector
of the present invention to the cell or population of cells to obtain a gene-
edited cell or
a population of gene-edited cells.
The gene-edited cell or population of gene-edited cells may be as defined
herein. The present
invention also provides a gene-edited cell or population of gene-edited cells
obtained or
obtainable by said method.
Step (a) providing a cell or a population of cells
The population of cells may be obtained or obtainable from any suitable
source. Suitably, the
population of cells are obtained or obtainable from (mobilised) peripheral
blood or cord blood.
The population of cells may be obtained or obtainable from a subject, e.g. a
subject to be
treated. Suitably, the population of cells may be isolated and/or enriched
from a biological
sample by any method known in the art, for example by FAGS and/or magnetic
bead sorting.
Suitably, the population of cells are mammalian cells, for example human
cells. The population
of cells may be, for example, autologous or allogeneic. The population of
cells may be, for
example, universal cells.
Suitably, the population of cells comprises about 1 x 105 cells per well to
about 10 x 105 cells
per well, e.g. about 2 x 105 cells per well, or about 5 x 105 cells per well.
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The population of cells may comprise HSCs, HPCs, and/or LPCs. Suitably, at
least 50%, at
least 60%, at least 70%, or at least 80% of the population of cells are HSCs,
HPCs, and/or
LPCs. In some embodiments, the population of cells consists essentially of
HSCs, HPCs,
and/or LPCs, or consists of HSCs, HPCs, and/or LPCs.
The population of cells may comprise CD34+ cells, e.g. CD34+ HSCs, HPCs,
and/or LPCs.
Suitably, at least 50%, at least 60%, at least 70%, or at least 80% of the
population of cells
are CD34+ cells, e.g. CD34+ HSCs, HPCs, and/or LPCs. In some embodiments, the
population of cells consists essentially of CD34+ cells, e.g. CD34+ HSCs,
HPCs, and/or LPCs,
or consists of CD34+ cells, e.g. CD34+ HSCs, HPCs, and/or LPCs.
The population of cells may comprise CD34+CD133+CD90+ cells, CD34+CD133+CD90-
cells, and/or CD34+CD133-CD90-. Suitably, at least 50%, at least 60%, at least
70%, or at
least 80% of the population of cells are 0D34+0D133+CD90+ cells,
0D34+0D133+CD90-
cells, and/or CD34+CD133-CD90- cells. In some embodiments, the population of
cells
consists essentially of CD34+CD133+CD90+ cells, CD34+CD133+CD90- cells, and/or
CD34+CD133-CD90- cells, or consists of CD34+CD133+CD90+ cells, CD34+CD133+CD90-
cells, and/or CD34+CD133-CD90- cells.
The cell or population of cells may be cultured prior to step (b). The pre-
culturing step may
comprise a pre-activation step and/or a pre-expansion step, optionally the pre-
culturing step
is a pre-activation step.
As used herein, a "pre-culturing step" refers to a culturing step which occurs
prior to genetic
modification of the cells. As used herein, a "pre-activating step" refers to
an activation step or
stimulation step which occurs prior to genetic modification of the cells. As
used herein, a "pre-
expansion step" refers to an expansion step which occurs prior to genetic
modification of the
cells.
Suitably, the method may comprise:
(al) providing a population of cells;
(a2) pre-culturing (e.g. pre-activating and/or pre-expanding) the population
of cells to
obtain a pre-cultured (e.g. pre-activated and/or pre-expanded) population of
cells;
(b) delivering an RNA-guided nuclease, a guide RNA, and/or a polynucleotide or
vector
of the present invention to the pre-cultured (e.g. pre-activated and/or pre-
expanded)
population of cells to obtain a population of gene-edited cells.
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The pre-culturing step (e.g. pre-activation step and/or pre-expansion step)
may be carried out
using any suitable conditions.
During the pre-culturing step (e.g. pre-activation step and/or pre-expansion
step) the
population of cells may be seeded at a concentration of about 1 x 105 cells/ml
to about 10 x
105 cells/ml, e.g. about 2 x 105 cells/ml, or about 5 x 105 cells/ml.
Suitably, the pre-culturing step (e.g. pre-activation step and/or pre-
expansion step) is at least
1 day, at least 2 days, or at least 3 days. Suitably, the population of cells
are pre-cultured (e.g.
pre-activated and/or pre-expanded) for about 3 days. Suitably, the population
of cells are pre-
cultured in a 5% CO2 humidified atmosphere at 37 C.
Any suitable culture medium may be used. For example, commercially available
medium such
as StemSpan medium may be used, which contains bovine serum albumin, insulin,
transferrin,
and supplements in Iscove's MDM. The culture medium may be supplemented with
one or
more antibiotic (e.g. penicillin, streptomycin).
The pre-culturing step (e.g. pre-activation step and/or pre-expansion step)
may be carried out
in the presence in of one or more cytokines and/or growth factors. As used
herein, a "cytokine"
is any cell signalling substance and includes chemokines, interferons,
interleukins,
lymphokines, and tumour necrosis factors. As used herein, a "growth factor" is
any substance
capable of stimulating cell proliferation, wound healing, or cellular
differentiation. The terms
"cytokine" and "growth factor' may overlap.
The pre-culturing step (e.g. pre-activation step and/or pre-expansion step)
may be carried out
in the presence of one or more early-acting cytokine, one or more transduction
enhancer,
and/or one or more expansion enhancer.
Early-acting cytokines
As used herein, an "early-acting cytokine" is a cytokine which stimulates
HSCs, HPCS, and/or
LPCs or CD34+ cells. Early-acting cytokines include thrombopoietin (TPO), stem
cell factor
(SCF), Flt3-ligand (FLT3-L), interleukin (IL)-3, and IL-6. In some
embodiments, the pre-
culturing step (e.g. pre-activation step and/or pre-expansion step) is carried
out in the
presence of at least one early-acting cytokine. Any suitable concentration of
early-acting
cytokine may be used. For example, 1-1000 ng/ml, or 10-1000 ng/ml, or 10-500
ng/ml.
In some embodiments, the pre-culturing step (e.g. pre-activation step and/or
pre-expansion
step) is carried out in the presence of SCF. The concentration of SCF may be
about 10-1000
ng/ml, about 50-500 ng/ml, or about 100-300 ng/ml.
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In some embodiments, the pre-culturing step (e.g. pre-activation step and/or
pre-expansion
step) is carried out in the presence of FLT3-L. The concentration of FLT3-L
may be about 10-
1000 ng/ml, about 50-500 ng/ml, or about 100-300 ng/ml.
In some embodiments, the pre-culturing step (e.g. pre-activation step and/or
pre-expansion
step) is carried out in the presence of TPO. The concentration of TPO may be
about 5-500
ng/ml, about 10-200 ng/ml, or about 20-100 ng/ml.
In some embodiments, the pre-culturing step (e.g. pre-activation step and/or
pre-expansion
step) is carried out in the presence of IL-3. The concentration of IL-3 may be
about 10-200
ng/ml, about 20-100 ng/ml, or about 60 ng/ml.
In some embodiments, the pre-culturing step (e.g. pre-activation step and/or
pre-expansion
step) is carried out in the presence of IL-6. The concentration of IL-6 may be
about 5-100
ng/ml, about 10-50 ng/ml, or about 20 ng/ml.
In some embodiments, the pre-culturing step (e.g. pre-activation step and/or
pre-expansion
step) is carried out in the presence of SCF (e.g. in a concentration of about
100 ng/ml), FLT3-
L (e.g. in a concentration of about 100 ng/ml), TPO (e.g. in a concentration
of about 20 ng/ml)
and IL-6 (e.g. in a concentration of about 20 ng/ml), in particular when the
population of cells
are cord-blood 0D34+ cells.
In some embodiments, the pre-culturing step (e.g. pre-activation step and/or
pre-expansion
step) is carried out in the presence of SCF (e.g. in a concentration of about
300 ng/ml), FLT3-
L (e.g. in a concentration of about 300 ng/ml), TPO (e.g. in a concentration
of about 100 ng/ml)
and IL-3 (e.g. in a concentration of about 60 ng/ml), in particular when the
population of cells
are (mobilised) peripheral blood CD34+ cells.
Transduction enhancers
As used herein, a "transduction enhancer' is a substance that is capable of
improving viral
transduction of HSCs, HPCS, and/or LPCs or CD34+ cells. Suitable transduction
enhancers
include LentiBOOST, prostaglandin E2 (PGE2), protamine sulfate (PS),
Vectofusin-1,
ViraDuctin, RetroNectin, staurosporine (Stauro), 7-hydroxy-stauro, human serum
albumin,
polyvinyl alcohol, and cyclosporin H (CsH). In some embodiments, the pre-
culturing step (e.g.
pre-activation step and/or pre-expansion step) is carried out in the presence
of at least one
transduction enhancer. Any suitable concentration of transduction enhancer may
be used, for
example as described in Schott, J.W., et al., 2019. Molecular Therapy-Methods
& Clinical
Development, 14, pp.134-147 or Yang, H., et al., 2020. Molecular Therapy-
Nucleic Acids, 20,
pp. 451-458.
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In some embodiments, the pre-culturing step (e.g. pre-activation step and/or
pre-expansion
step) is carried out in the presence of PGE2. Suitably, the PGE2 is 16,16-
dimethyl
prostaglandin E2 (dmPGE2). The concentration of PGE2 may be about 1-100 pM,
about 5-20
pM, or about 10 pM.
In some embodiments, the pre-culturing step (e.g. pre-activation step and/or
pre-expansion
step) is carried out in the presence of CsH. The concentration of CsH may be
about 1-50 pM,
5-50 pM, about 10-50 pM, or about 10 pM.
Expansion enhancers
As used herein, an "expansion enhancer" is a substance that is capable of
improving
expansion of HSCs, HPCS, and/or LPCs or CD34+ cells. Suitable expansion
enhancers
include UM171, UM729, StemRegenin1 (SR1), diethylaminobenzaldehyde (DEAB),
LG1506,
BIO (GSK3[3 inhibitor), NR-101, trichostatin A (TSA), garcinol (GAR), valproic
acid (VPA),
copper chelator, tetraethylenepentamine, and nicotinamide. In some
embodiments, the pre-
culturing step (e.g. pre-activation step and/or pre-expansion step) is carried
out in the
presence of at least one expansion enhancer. Any suitable concentration of
expansion
enhancer may be used, for example as described in Huang, X., et al., 2019.
F1000Research,
8, 1833.
In some embodiments, the pre-culturing step (e.g. pre-activation step and/or
pre-expansion
step) is carried out in the presence of UM171 or UM729. The concentration of
UM171 may be
about 10-200 nM, about 20-100 nM, or about 50 nM.
In some embodiments, the pre-culturing step (e.g. pre-activation step and/or
pre-expansion
step) is carried out in the presence of SRI. The concentration of SRI may be
about 0.1-10
pM, about 0.5-5 pM, or about 1 pM.
In some embodiments, the pre-culturing step (e.g. pre-activation step and/or
pre-expansion
step) is carried out in the presence of UM171 (e.g. in a concentration of
about 50 nM) or
UM729 and SR1 (e.g. in a concentration of about 1 pM).
In some embodiments, the pre-culturing step (e.g. pre-activation step and/or
pre-expansion
step) is carried out in the presence of SCF (e.g. in a concentration of about
100 ng/ml), FLT3-
L (e.g. in a concentration of about 100 ng/ml), TPO (e.g. in a concentration
of about 20 ng/ml),
IL-6 (e.g. in a concentration of about 20 ng/ml), PGE2 (e.g. in a
concentration of about 10 pM),
UM171 (e.g. in a concentration of about 50 nM), and SR1 (e.g. in a
concentration of about 1
pM), in particular when the population of cells are cord-blood CD34+ cells.
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In some embodiments, the pre-culturing step (e.g. pre-activation step and/or
pre-expansion
step) is carried out in the presence of SCF (e.g. in a concentration of about
300 ng/ml), FLT3-
L (e.g. in a concentration of about 300 ng/ml), TPO (e.g. in a concentration
of about 100 ng/ml),
IL-3 (e.g. in a concentration of about 60 ng/ml), PGE2 (e.g. in a
concentration of about 10 pM),
UM171 (e.g. in a concentration of about 50 nM), and SR1 (e.g. in a
concentration of about 1
pM), in particular when the population of cells are (mobilised) peripheral
blood CD34+ cells.
Step (b) obtaining a gene-edited cell or a population of gene-edited cells
A kit, composition, and/or gene-editing system comprising an RNA-guided
nuclease, a guide
RNA, and/or a polynucleotide or vector of the present invention may, for
example, be used to
obtain the gene-edited cell or a population of gene-edited cells.
The RNA-guided nuclease, guide RNA, and/or polynucleotide or vector may be any
suitable
combination described herein. The guide RNA may correspond to the same DSB
site targeted
by the homology arms. The RNA-guided nuclease may correspond to the guide RNA
used.
For example:
(i) the RNA-guided nuclease may be a Cas9 endonuclease;
(ii) the guide RNA may be a guide RNA comprising or consisting of a nucleotide
sequence that has at least 90% identity or at least 95% identity to any of SEQ
ID NOs:
41-52 or 53-55, optionally wherein the guide RNA comprises or consists of a
nucleotide
sequence that has at least 90% identity or at least 95% identity to SEQ ID NO:
41 or
53 (preferably SEQ ID NO: 41); and
(iii) the polynucleotide may be a polynucleotide comprising from 5' to 3': a
first
homology region, a splice acceptor sequence, a nucleotide sequence encoding a
RAG1 polypeptide, and a second homology region, wherein the first homology
region
comprises or consists of a nucleotide sequence that has at least 70% identity,
at least
80% identity, at least 90% identity, at least 95% identity, at least 98%
identity, or 100%
identity to any of SEQ ID NOs: 7-18 and/or the second homology region
comprises or
consists of a nucleotide sequence that has at least 70% identity, at least 80%
identity,
at least 90% identity, at least 95% identity, at least 98% identity, or 100%
identity to
any of SEQ ID NOs: 19-30; or the vector may be a vector comprising said
polynucleotide.
In some embodiments:
(i) the RNA-guided nuclease may be a Cas9 endonuclease;
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(ii) the guide RNA comprises or consists of a nucleotide sequence that has at
least
90% identity, at least 95% identity or 100% identity to SEQ ID NO: 41 or 53
(preferably
SEQ ID NO: 41); and
(iii) the polynucleotide comprises from 5' to 3': a first homology region, a
splice
acceptor sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a
second homology region, wherein the first homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90%
identity, at least 95% identity, at least 98% identity, or 100% identity to
SEQ ID NO:
31, or a fragment thereof; and the second homology region comprises or
consists of a
nucleotide sequence that has at least 70% identity, at least 80% identity, at
least 90%
identity, at least 95% identity, at least 98% identity, or 100% identity to
SEQ ID NO:
32, or a fragment thereof; or the vector comprises said polynucleotide.
Delivery of a RNA-guided nuclease, guide RNA, and/or polynucleotide or vector
The RNA-guided nuclease, guide RNA, and/or polynucleotide or vector may be
delivered to
the cell by any suitable technique. For example, the RNA-guided nuclease may
be delivered
directly using electroporation, microinjection, bead loading or the like, or
indirectly via
transfection and/or transduction. The guide RNA, and/or polynucleotide or
vector may be
introduced by transfection and/or transduction.
As used herein "transfection" is a process using a non-viral vector to deliver
a polypeptide
and/or polynucleotide to a target cell. Typical transfection methods include
electroporation,
DNA biolistics, lipid-mediated transfection, compacted DNA-mediated
transfection, liposomes,
immunoliposomes, lipofectin, cationic agent-mediated transfection, cationic
facial amphiphiles
(CFAs) and combinations thereof.
As used herein "transduction" is a process using a viral vector to deliver a
polynucleotide to a
target cell. Typical transduction methods include infection with recombinant
viral vectors, such
as adeno-associated viral, retroviral, lentiviral, adenoviral, baculoviral and
herpes simplex viral
vectors.
The RNA-guided nuclease and the guide RNA may be delivered by any suitable
method, for
instance any method described in Wilbie, D., et al., 2019. Accounts of
chemical research,
52(6), pp.1555-1564. Suitably, the RNA-guided nuclease and the guide RNA are
delivered
together preassembled as in the form of a RNP complex. The RNP complex may be
delivered
by electroporation.
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Any suitable dose of the RNA-guided nuclease and/or the guide RNA may be used.
For
example, the guide RNA may be delivered at a dose of about 10-100 pmol/well,
optionally
about 50 pmol/well. For example, the RNP may be delivered at a dose of about 1-
10 pM,
optionally 1-2.5 pM.
The RNA-guided nuclease and/or the guide RNA may be delivered prior to the
vector and/or
simultaneously with the polynucleotide or vector of the invention. Suitably,
the RNA-guided
nuclease and/or the guide RNA are delivered prior to the polynucleotide or
vector. For
example, the RNA-guided nuclease and/or the guide RNA may be delivered about 1-
100
minutes, about 5-30, or about 15 minutes, prior to the polynucleotide or
vector.
The polynucleotide or vector of the invention may be delivered by any suitable
method. For
example, when the polynucleotide may be in a viral vector or the vector may be
a viral vector
and delivered by transduction.
Any suitable dose of the polynucleotide or vector may be used. For example,
the vector may
be delivered at a MOI of about 104 to 105 vg/cell, optionally about 104
vg/cell.
Delivery of a p53 inhibitor and/or HDR enhancer
The method may further comprise a step of delivering a p53 inhibitor and/or
HDR enhancer.
The p53 inhibitor and/or HDR enhancer may be delivered simultaneously. The p53
inhibitor
and/or HDR enhancer may be delivered simultaneously with or after the RNA-
guided nuclease
and/or the guide RNA.
As used herein, a "p53 inhibitor" is a substance which inhibits activation of
the p53 pathway.
The p53 pathway plays a role in regulation or progression through the cell
cycle, apoptosis,
and genomic stability by means of several mechanisms including: activation of
DNA repair
proteins, arrest of the cell cycle; and initiation of apoptosis. Inhibition of
this p53 response by
delivery during editing has been shown to increase hematopoietic repopulation
by treated cells
(Schiroli, G. et al. 2019. Cell Stem Cell 24, 551-565). Suitably, the p53
inhibitors is a dominant-
negative p53 mutant protein, e.g. GSE56.
GSE56 may have the amino acid sequence:
CPGRDRRTEEEN FRKKEEHCPELPPGSAKRALPTSTSSSPQQKKKPLDGEYFTLKI RG
RERFEM FRELNEALELKDARAAEESGDSRAHSSYPK
(SEQ ID NO: 67)
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In one embodiment, the p53 dominant negative peptide is a variant of GSE56
comprising 1,
2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions or deletions,
while retaining the
activity of GSE56, for example in reducing or preventing p53 signalling.
In one embodiment, the p53 dominant negative peptide comprises an amino acid
sequence
having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to
SEQ ID
NO: 67.
As used herein, an "HDR enhancer" is a substance that is capable of improving
HDR efficiency
in HSCs, HPCS, and/or LPCs or CD34+ cells. HDR is constrained in long-term-
repopulating
HSCs. Any suitable HDR enhancer may be used, for example as described in
Ferrari, S., et
al., 2020. Nature Biotechnology, pp.1-11. Suitably, the HDR enhancer is the
adenovirus 5
E4orf6/7 protein. Adenovirus 5 E4orf6/7 proteins may be as disclosed in WO
2020/002380
(incorporated herein by reference).
The p53 inhibitor and the HDR enhancer may be delivered by any suitable
method. The p53
inhibitor and/or the HDR enhancer may be transiently expressed, for example
the p53 inhibitor
and/or the HDR enhancer may delivered via mRNA. The p53 inhibitor and the HDR
enhancer
may be delivered by separate mRNAs or on a single mRNA encoding a fusion
protein,
optionally with a self-cleaving peptide (e.g. P2A). Any suitable dose of the
p53 inhibitor and/or
the HDR enhancer may be used, for example mRNA be delivered at a concentration
of about
10-1000 pg/ml, about 50-500 pg/ml, or about 150 pg/ml.
In some embodiments, step (b) comprises:
(b1) delivering a RNA-guided nuclease and a guide RNA of the invention,
optionally
preassem bled in the form of a RNP complex by electroporation;
(b2) optionally, delivering a p53 inhibitor and/or a HDR enhancer; and
(b3) delivering a polynucleotide or vector of the invention by transduction to
provide a
gene-edited population of cells.
Culturing the gene-edited cell or population of gene-edited cells
The method may further comprise a step of culturing the population of gene-
edited cells. This
may be an expansion step, i.e. the method may further comprises a step of
expanding the
population of gene-edited cells.
The culturing step (e.g. expansion step) may be carried out using any suitable
conditions.
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During the culturing step (e.g. expansion step) the population of cells may be
seeded at a
concentration of about 1 x 105 cells/ml to about 10 x 105 cells/ml, e.g. about
2 x 105 cells/ml,
or about 5 x 105 cells/ml. Suitably, the culturing step (e.g. expansion step)
is for at least one
day, or one to five days. For example, the culturing step (e.g. expansion
step) may be for
about one day. Suitably, the population of cells are cultured in a 5% CO2
humidified
atmosphere at 37 C.
Any suitable culture medium may be used. For example, commercially available
medium such
as StemSpan medium may be used, which contains bovine serum albumin, insulin,
transferrin,
and supplements in Iscove's MDM. The culture medium may be supplemented with
one or
more antibiotic (e.g. penicillin, streptomycin). The culturing step (e.g.
expansion step) may be
carried out in the presence in of one or more cytokines and/or growth factors.
In some embodiments, step (b) comprises:
(b1) delivering a RNA-guided nuclease and a guide RNA of the invention,
optionally
preassembled in the form of a RNP complex by electroporation;
(b2) optionally, delivering a p53 inhibitor and/or a HDR enhancer;
(b3) delivering a polynucleotide or vector of the invention by transduction to
provide a
gene-edited population of cells; and
(b4) culturing (e.g. expanding) the gene-edited population of cells.
Methods of treatment
In one aspect the present invention provides a method of treating a subject
using
polynucleotides, vectors, guide RNAs, kits, compositions, gene-editing
systems, cells and/or
populations of cells of the present invention. Suitably, the method of
treating a subject may
comprise administering a cell or population of cells of the present the
invention.
In a related aspect the present invention provides a polynucleotide, vector,
guide RNA, kit,
composition, gene-editing system, cell and/or populations of cells of the
present invention for
use as a medicament. Suitably, the cell or population of cells of the present
the invention may
be used as a medicament.
In a related aspect, the present invention provides use of a polynucleotide,
vector, guide RNA,
kit, composition, gene-editing system, cell and/or populations of cells of the
present invention
for the manufacture of a medicament. Suitably, the cell or population of cells
of the present
the invention may be used for the manufacture of a medicament.
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Suitably, a method of treating a subject may comprise:
(a) providing a cell or a population of cells;
(b) using a kit, composition, and/or gene-editing system described herein to
obtain a
gene-edited cell or a population of gene-edited cells; and
(c) administering the population of gene-edited cells to the subject.
For example, a method of treating a subject may comprise:
(a) providing a cell or a population of cells;
(b) delivering an RNA-guided nuclease, a guide RNA, and/or a polynucleotide or
vector
of the present invention to the cell or population of cells to obtain a gene-
edited cell or
a population of gene-edited cells; and
(c) administering the population of gene-edited cells to the subject.
Steps (a) and (b) may be identical to the steps described in the section
above.
Suitably, the cell of population of cells may be isolated and/or enriched from
the subject to be
treated, e.g. the population of cells may be an autologous population of CD34+
cells. Suitably,
the population of cells are isolated from (mobilised) peripheral blood or cord
blood of the
subject to be treated and subsequently enriched (e.g. by FACS and/or magnetic
bead soiling).
The subject may be immunocompromised and/or the disease to be treated may be
an
immunodeficiency, i.e the medicament may be for treating an immunodeficiency.
As used
herein, an "immunodeficiency" is a disease in which the immune system's
ability to fight
infectious disease and cancer is compromised or entirely absent. A subject who
has an
immunodeficiency is said to be "immunocompromised". An immunocompromised
person may
be particularly vulnerable to opportunistic infections, in addition to normal
infections that could
affect everyone.
RAG deficient-immunodeficiency
The subject may have RAG deficiency, e.g. a RAG1 deficiency. A RAG1 deficiency
may be
due to a loss-of-function mutation in the RAG1 gene, optionally a loss-of-
function mutation in
the RAG1 exon 2.
The immunodeficiency may be a RAG deficient-immunodeficiency. As used herein,
a "RAG
deficient-immunodeficiency" is an immunodeficiency characterised by loss of
RAG1/RAG2
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activity. A RAG deficient-immunodeficiency may, for example be caused by a
mutation in RAG
genes.
Suitably, the RAG deficient-immunodeficiency may be a RAG1 deficiency. A RAG1
deficiency
may be due to a loss-of-function mutation in the RAG1 gene, optionally a loss-
of-function
mutation in the RAG1 exon 2.
Mutations of the RAG genes in humans are associated with distinct clinical
phenotypes, which
are characterized by variable association of infections and autoimmunity. In
some cases,
environmental factors have been shown to contribute to such phenotypic
heterogeneity. In
humans, RAG1 deficiency can cause a broad spectrum of phenotypes, including T-
B- SCID,
Omenn syndrome (OS), atypical SCID (AS) and combined immunodeficiency with
granuloma/autoimmunity (CID-G/AI). (Notarangelo, L.D., et al., 2016. Nature
Reviews
Immunology, 16(4), pp.234-246 and Delmonte, 0.M., et al., 2018. Journal of
clinical
immunology, 38(6), pp.646-655).
In some embodiments, the RAG deficient-immunodeficiency is T- B- SCID, Omenn
syndrome,
atypical SCID, or CID-G/Al.
Severe combined immunodeficiency (SCID) comprises a heterogeneous group of
disorders
that are characterized by profound abnormalities in the development and
function of T cells
(and also B cells in some forms of SCID), and are associated with early-onset
severe
infections. This condition is inevitably fatal early in life, unless immune
reconstitution is
achieved, usually with HSCT. Following the introduction of newborn screening
for SCID in the
United States, it has become possible to establish that RAG mutations account
for 19% of all
cases of SCID and SCID-related conditions, and are a prominent cause of
atypical SCID and
Omenn syndrome in particular. (Notarangelo, L.D., et al., 2016. Nature Reviews
Immunology,
16(4), pp.234-246).
In 1996, RAG mutations were identified as the main cause of T-B- SCID with
normal cellular
radiosensitivity. A distinct phenotype characterizes Omenn syndrome, which was
first
described in 1965. These patients manifest early-onset generalized
erythroderma,
lymphadenopathy, hepatosplenomegaly, eosinophilia and severe
hypogammaglobulinaemia
with increased IgE levels, which are associated with the presence of
autologous, oligoclonal
and activated T cells that infiltrate multiple organs. In some patients with
hypomorphic RAG
mutations, a residual presence of autologous T cells was demonstrated without
clinical
manifestations of Omenn syndrome. This condition is referred to as 'atypical'
or 'leaky' SCID.
A distinct SCID phenotype involving the oligoclonal expansion of autologous y5
T cells
(referred to here as y51 T+ SCID) has been reported in infants with RAG
deficiency and
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disseminated cytomegalovirus (CMV) infection. (Notarangelo, L.D., et al.,
2016. Nature
Reviews Immunology, 16(4), pp.234-246).
Whereas SCID, atypical SCID and Omenn syndrome are inevitably fatal early in
life if
untreated, several forms of RAG deficiency with a milder clinical course and
delayed
presentation have been reported in recent years. In particular, the occurrence
of CID¨G/AI
was reported in three unrelated girls with RAG mutations who manifested
granulomas in the
skin, mucous membranes and internal organs, and had severe complications after
viral
infections, including B cell lymphoma. Following this description, several
other cases of CI D¨
G/AI with various autoimmune manifestations (such as cytopaenias, vitiligo,
psoriasis,
myasthenia gravis and Guillain¨Barre syndrome) have been reported.
(Notarangelo, L.D., et
al., 2016. Nature Reviews Immunology, 16(4), pp.234-246).
Additional phenotypes that are associated with RAG deficiency include
idiopathic CD4+ T cell
lymphopaenia, common variable immunodeficiency, IgA deficiency, selective
deficiency of
polysaccharide-specific antibody responses, hyper-IgM syndrome and sterile
chronic
multifocal osteomyelitis. (Notarangelo, L.D., et al., 2016. Nature Reviews
Immunology, 16(4),
pp.234-246).
The skilled person will understand that they can combine all features of the
invention disclosed
herein without departing from the scope of the invention as disclosed.
Preferred features and embodiments of the invention will now be described by
way of non-
limiting examples.
The practice of the present invention will employ, unless otherwise indicated,
conventional
techniques of chemistry, biochemistry, molecular biology, microbiology and
immunology,
which are within the capabilities of a person of ordinary skill in the art.
Such techniques are
explained in the literature. See, for example, Sambrook, J., Fritsch, E.F. and
Maniatis, T.
(1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor
Laboratory
Press; Ausubel, F.M. et al. (1995 and periodic supplements) Current Protocols
in Molecular
Biology, Ch. 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn,
A. (1996) DNA
Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J.M.
and McGee,
J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford
University Press; Gait, M.J.
(1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and LiIley,
D.M. and
Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis
and Physical
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Analysis of DNA, Academic Press. Each of these general texts is herein
incorporated by
reference.
EXAMPLES
EXAMPLE 1 ¨ Editing the RAGI gene
Results
We have developed a platform to correct CD34+ hematopoietic stem cells by
exploiting a gene
targeting approach.
In the approach described herein, we deliver by nucleofection a Cas9
ribonucleoprotein (RN F)
that introduces a DNA double strand break (DSB) in the first intron of RAG1
gene. Following
the DNA DSB, the corrective donor DNA, delivered by AAV6 vector, is integrated
by homology
directed repair (HDR), thanks to the presence of two sequences, flanking the
corrective donor,
that are homologous to the Cas9 cutting site. An alternative splicing acceptor
(SA) upstream
of the corrective DNA allows the endogenous promoter of RAG1 to control the
expression of
the transgene (Figure 1 panel A). Of note, RAG1 exon 2 contains the whole
coding sequence,
thus integrating a corrective RAG1 coding sequence upstream of exon 2 may be
therapeutic
for any RAG1 mutation with clinical relevance.
Generation of NALM6 and K562 Cas9 Cell lines
First, to test our panel of Cas9 guide RNAs we generated two cell lines with
inducible Cas9
expression. NALM6 and K562 cell lines were transduced with a lentiviral vector
carrying the
Cas9 cassette under the control of a TET-inducible promoter and a cassette
that confers
resistance to puromycin. After transduction with MOI 20 the two cell lines
were kept in culture
with puromycin 1.5 pg/ml for one week to select the transduced cells (Figure 1
panel B). After
puromycin selection, a VCN 3.65 and a VCN 4.35 were verified by LTR specific
ddPCR in
NALM6 Cas9 and K562 Cas9 cell line respectively (Figure 1 panel C). Efficient
Cas9
expression was also verified by RT-qPCR after two days of induction with
scaling doses of
doxycycline (Figure 1 panel D). The highest Cas9 expression was found at the
dose of 1
pg/ml of doxycyclin in both the cell lines.
RAG1 Guide Selection
A panel of nine guides was first identified to target three non-repeated loci
of RAG1 intron 1.
In addition, three guides (gRNA 1,2,3) targeting the first 200 bp of RAG1 exon
2 were designed
with the final aim to integrate the corrective RAG1 coding sequence in frame
with the
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endogenous ATG. This strategy would exploit the endogenous splice acceptor
thus preserving
any putative endogenous splicing regulations (Figure 2 A).
Guides were electroporated as plasmid DNAs in K562 Cas9 and NALM6 Cas9 cell
lines
considering two different doses (10Ong/well and 200ng/well.) Cas9 expression
was induced
the day before the electroporation and for the two following days by adding
doxycycline (1
pg/ml) to the medium. Genomic DNA was extracted at day 7 and cutting frequency
was
evaluated measuring the percentage of NHEJ-mediated indel mutations by 17
nuclease assay
(scheme shown in Figure 2 B).
The majority of the tested guides had good cutting frequency showing similar
results in both
cell lines. In particular, Guide 9 was the best performing guide targeting the
intron with a cutting
frequency up to 72.7% in K562 Cas9 and 78.5% in NALM6 Cas9. Similar cutting
frequencies
were also achieved by Guide 7, that showed a cutting frequency up to 67.5% in
K562 Cas9
and 70.5% in NALM6 Cas9 cell lines. Guide 3 was the best performing guide
targeting the
exon with a cutting frequency up to 58.9% in K562 Cas9 (Figure 2 C) and 73.5%
in NALM6
Cas9 (Figure 2 D). Of note, despite the higher expression of Cas9 expression
in K562 Cas9
than in NALM6 Cas9 cell line, no difference in the overall cutting efficiency
was observed.
Cutting frequency was also tested in NALM6 VVT using in vitro preassemble RNP
of guide 9
and guide 3 at the dose of 25 or 50 pmol/well (Figure 2 E). Both guides
retained a good
activity, guide 3 reached up to 71.5% cutting frequency and guide 9 up to
78.5% at the higher
dose of RN P.
Donor DNA Optimization
Guide 9 was further tested in NALM6 Cas9 and K562 Cas9 cell lines to verify
the correct
integration of the PGK_GFP reporter cassette flanked by two homology arms.
We also assessed the ability of the endogenous RAG1 promoter to induce the
expression of
the GFP in the absence of the PGK promoter using a donor plasmid containing
splice acceptor
(SA) SA_GFP cassette. RAG1 expression occurs only during lymphocytes
differentiation at
DN2 T and pro-B cell stages. To assess whether the endogenous promoter of RAG1
was able
to induce the expression of the GFP cassette, we exploited NALM6 cell line, a
Pre-B cell line
that constitutively expresses RAG1 (Figure 3 A). As mentioned, RAG1 genomic
region is
composed of two exons and the whole coding sequence, which is 3.1 Kb, is
encoded by the
second exon, followed by a long 3'UTR region of 3.3 Kb. Our correction
strategy plans to
deliver an AAV6 vector containing the entire coding sequence targeting the
intronic region
upstream of exon 2. The 3'UTR region (>3 Kb) downstream of the RAG1 coding
sequence
was not inserted because of the limited size hosted by the AAV6 vector.
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To assess whether the 3'UTR of RAG1 is necessary for the efficient expression
of our
corrective donor, we generated four different SA_GFP donor DNAs (Figure 3 B):
construct carrying the bovine growth hormone (BGH) PolyA downstream the
SA_GFP (SA_GFP_BGH);
ii.
construct carrying the Woodchuck hepatitis virus post-transcriptional
regulatory
element (WPRE) downstream the SA_GFP and upstream the BGH PolyA
(SA_GFP_WPRE). WPRE has been reported to generally enhance transgene
expression;
construct with the same endogenous RAG1 3'UTR following the SA_GFP
cassette (SA_GFP_3'UTR);
iv.
construct containing a splice donor downstream the SA_GFP cassette
(SA_GFP_SD) to obtain a fusion transcript including the corrected sequence and
endogenous RAG1 followed by the 3' UTR sequence (Figure 3 C).
NALM6 Cas 9 and K562 Cas9 cell lines, previously stimulated with doxycycline
to induce
Cas9, were transfected with guide 9 plasmid DNA (10Ong/well) and of various
linearized DNA
donors (1600ng/well). Stable integration of the donor DNA was verified by flow
cytometry as
GFP expression.
The PGK_GFP positive control was stably integrated in both cell lines. In
particular, ten days
after transfection, 14% K562 Cas9 and 1.8% of NALM6 Cas9 were GFP positive
(Figure 3
D). Of note NALM6 cell line is particularly tricky to edit and we expected a
lower efficiency as
compared K562. Similar frequencies of GFP+ cells were observed in NALM6 Cas9
transfected
with the different SA_GFP donors, while almost no GFP + cells were detectable
in the K562
cell lines transfected with the SA_GFP donors. This observation confirms that
the endogenous
RAG1 promoter efficiently induces the expression of the SA_GFP cassette in the
NALM6 Cas9
cell line. Of note, the absence of GFP + cells in K562 Cas9 cell line, which
lacks RAG1
expression, further confirms that the GFP expression observed in NALM6 is
specifically
dependent on RAG1 promoter activity.
The effect of constructs carrying different 3'UTR was evaluated in NALM6 Cas9
cell line by
fluorescence intensity (MFI) of GFP + events at flow cytometry. The analysis
suggested that
the endogenous RAG1 3'UTR negatively affects the expression of the transgene.
GFP MFI
obtained upon transfection with SA_GFP_SD and SA_GFP_3'UTR constructs was
significantly lower than MFI obtained by SA_GFP_BGH (Figure 3 E, F). No
improvement was
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noticed using SA_GFP_ WPRE. Based on these data reporting GFP expression
level, we
decided to clone our vectors with the BGH_PolyA.
Off-target analysis
Preliminary in silico analysis demonstrated a promising off-target profile of
guide 9 and showed
that most likely off-targets fall in intronic regions thus suggesting a low
risk of off-target related
gene disruption events (Figure 4 A). A deeper characterization of the off-
target profile of guide
7 and 9 was pursued by an unbiased off-target detection assay (GUIDE-seq, Tsai
SO, et al.
Nat Biotechnol. 2015;33(2)1 87-97). The analysis was performed using 50 pmol
of High
Fidelity Cas9 Nuclease V3 on K562 cells resulting in 45.3% and 64.6% cutting
frequency by
guide 7 and 9, respectively (Figure 4 B). We achieved low (8.4%) ODN
integration for guide
7, but good frequency of integration for the guide 9 (38.2%) allowing the
analysis of off-target
in the samples (Figure 4 C). According to the analysis performed using the R
Bioconductor
package GUIDE-seq (Zhu LJ, et al. BMC Genomics. 2017;18(1)) using default
parameters,
no off-target site was identified for both guides. To deepen the investigation
also to very weak
potential off-targets, a second analysis with relaxed constraints was
performed, and two off-
target sites were found only for guide 7. These off-target sites fall into
intronic or intergenic
regions, with a number of mismatches >9 and at low frequency, indicating the
low risk profile
of guide 7. It is worth noting that no off-target sites were identified for
Guide 9.
Optimization of the gene editing protocol on human Cord blood-CD34+ cells
The editing procedure was then optimized in human CD34+ cells from cord blood
(hCB-CD34).
To this end, hCB-CD34 cells were thawed at day 0 and prestimulated for three
days seeding
1x106 cells/ml in StemSpan enriched with cytokines (hTPO 20ng/ml, hIL6
20ng/ml, hSCF
10Ong/ml, hFlt3-L 10Ong/ml, SRI 1uM, UM171 50nM).
At day 3, guides 3 and 9 were delivered by electroporation as in vitro
preassembled RNPs
and two doses were considered 25 and 50 pmol/well. To enhance cellular
stability, chemical
modification consisting in 2'-0-methyl 3'phosphorothioate were added at the
last three terminal
nucleotides at 5' and 3' ends of the guide RNAs. After 15', AAV6 vectors were
added to the
medium using three (104, 5x104, 106) MOI doses (Figure 5 A). To easily track
edited cells
using flow cytometry approach, two AAV6 donors (one for each guide) were used,
carrying
the PGK_GFP_BGH cassette flanked by two arms homologous to each of the two
cutting
sites. The toxicity of the procedure was assessed 24 hours after the
treatment, by staining the
cells with 7AAD and Annexin V and measuring the fraction of necrotic and
apoptotic cells by
flow cytometry. Four days after electroporation, we performed multiparametric
flow cytometry
analysis to evaluate the composition of various cellular subpopulations
composing the bulk
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treated cell culture and measure the percentage of GFP+ cells within these
subpopulations.
For this analysis, we took advantage of surface markers that allow identifying
the primitive
(CD34+CD133+CD90+), early (CD34+CD133+CD90-) and more committed (CD34+CD133-
CD90-) progenitors (Figure 5 B). Moreover, genomic DNA was extracted to
determine the
activity of the nucleases by T7 nuclease assay.
Guide 9 retained an activity comparable to that verified in NALM6 and K562
cell lines, 73.9%
cutting frequency was observed with 25pmol/well and 80.1% with 50pmol/well.
Guide 3
displayed a lower activity in hCB-CD34 with a cutting frequency of 16.9% and
19.3% with 25
and 50pm01/well respectively (Figure 5 C). In line with the latter
observation, targeted
integration with guide 3 was less efficient and at the dose of 25pm01/well,
with the highest MOI
(105), levels of integration were 18.3% in the bulk CD34+ and 1.25% in the
most primitive
subpopulation (Figure 5 D, E).
Guide 9 promoted a highly efficient targeted integration of the PGK_GFP
cassette. Apoptosis
analysis showed a low toxicity associated with the editing procedure, and
viability (7AAD-
AnnexinV- cells) was above 70% the day after the editing for all the
conditions tested (Figure
6 A). The analysis also suggested that AAV6 transduction had a stronger impact
on cell
viability than Cas9 transfection. In line with this observation, AAV6
transduction with MOI 105
impaired cell growth more than the transfection with 25pm01 Cas9, suggesting
that cell fitness
may be affected (Figure 6 B). High frequency of CD34+ cells (87.5%) in edited
conditions was
comparable to the untreated control (Figure 6 C). No major differences were
observed in
distribution of the three CD34+ subpopulations among different conditions
(Figure 6 D).
Analysis of integration frequency showed that that the most primitive
subpopulation
(CD34+CD133+CD90+) was the less permissive fraction. The highest editing
frequency in this
subpopulation was obtained using 25pm01 of Cas9 and the MOI 105 (52.8%). At
lower MOI,
the higher Cas9 dose (50pm01) enhanced the editing efficiency particularly in
the most
primitive subpopulation, indeed, with a MOI 104, editing frequency was 24.6%
and 40.5% with
25 and 50 pmol of Cas9 respectively (Figure 6 E). To confirm at molecular
level, the
integration observed by flow cytometry, genomic DNA was analysed by a ddPCR
assay using
a set of primers specific for the on-target integration. The percentages of
GFP measured by
flow cytometry and the percentages of HDR obtained with the ddPCR were
comparable, thus
corroborating that most integrations were on-target (Figure 6 F).
Overall, these data suggest that using this platform, we were able to obtain
efficient targeting
even in the most primitive CD34+ subpopulation. The editing protocol does not
affect the
phenotype of the cells (both in terms of total CD34+ cells and in terms of
subpopulation
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distribution). In particular, we identified a guide RNA promoting high
frequency of targeted
integration and set up editing conditions (50pm01/well Cas9 and MOI 104
Vg/cell) that allow
the best compromise between toxicity and targeting frequency (Figure 6 G).
In vivo transplantation of gene edited hCB-CD34+ cells
In order to assess if our procedure allows targeted integration in HSCs while
preserving their
long-term repopulating activity, edited CD34" cells were transplanted into
sublethally irradiated
NOD-scid IL2Rgnull mice (NSG) mice. Following the same protocol used in the
previous
experiment, after 3 days of stimulation, hCB-CD34+ cells were electroporated
with 50pmo1/well
of guide 9 RNP and 15 minutes later transduced with AAV6 at MOI 104 Vg/cell.
In this
experiment two distinct AAV6 vectors were used. The first AAV6 vector carrying
the
PGK_GFP_BGH was used as a positive control to easily follow engraftment of
edited cells.
The second donor carrying a SA_GFP_BGH was used to assess the in vivo
expression of
GFP gene under the control of RAG1 endogenous promoter. The day following the
editing
procedure, treated hCB-CD34+ 350,000 cells/mouse were injected in 4-5 NSG mice
per group,
6 hours after sublethal total body irradiation (120 rad). In order to assess
the levels of gene
targeting efficiency after the treatment, few cells were maintained in culture
for 4 more days.
Using both the AAV6 vectors we measured -80% of targeted integration by ddPCR
(Figure 7
A), thus recapitulating the results obtained in the previous experiments. Flow
cytometric
analysis of the peripheral blood obtained from transplanted mice was performed
6, 9, 13
weeks after transplantation and at sacrifice at 17 weeks. Analysis of
frequency of hCD45" cells
on total live cells in peripheral blood confirmed that treated cells were
present at normal levels
(up to -56%), suggesting long-term engraftment, and with a similar kinetics in
the two groups
(Figure 7 B). With regard to peripheral blood composition, mice showed no
major skew in the
subpopulation composition and a normal presence of B, T and myeloid cells in
both the groups
confirming that the editing procedure does not affect multi-lineage
differentiation (Figure 7 D,
F, H).
In the group of mice receiving cells treated with PGK_GFP_BGH vector, edited
hCD45+ GFP+
cells were maintained over time at high percentage (-40-50%), thus suggesting
that the
treatment was tolerated from the most primitive cells and confirming their
long-term survival
in vivo (Figure 7 C). Similar levels of edited hCD45" GFP + cells were found
among B cells, T
cells and myeloid cells in peripheral blood, confirming that edited cells
maintained multilineage
differentiation capacity (Figure 7 E, G, I). In mice transplanted with
SA_GFP_BGH treated
cells, despite the efficient targeting frequency observed in vitro, we
observed a reduced
frequency of GFP+ cells in peripheral blood (Figure 7 C). Myeloid and
circulating T cells were
GFP negative, as expected, because these two cell populations do not express
RAG1 (Figure
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7 G, I). Conversely, relevant percentage (-18%) of GFP + cells was observed
among circulating
B cells (Figure 7 E) likely due to their immature phenotype as the majority of
B cells expressed
CD24 and CD38.
At sacrifice, analysis of the bone marrow confirmed the engraftment of treated
CD34+ stem
cells. Moreover, in the PGK_GFP_BGH group, a high frequency of GFP + targeted
cells (-38%)
was observed among the CD34+ cells further suggesting efficient engraftment of
long-term
repopulating stem cells (Figure 7 L and M). Although the thymus in NSG mice is
atrophic and
dysfunctional, we analyzed GFP expression during thymopoiesis according to CD4
and CD8
expression (Figure 7 N). With the PGK_GFP_BGH cassette GFP expression was
uniform
among the developmental stages and no differences were observed between
immature
thymocytes and mature circulating T cells. Conversely, using SA_GFP_BGH
cassette as
donor, GFP expression was found in developing thymocytes, while almost no GFP
expression
was detected in peripheral blood and splenic T cells (Figure 7 N).
Taken together these observations suggest that we have established an
efficient protocol for
the editing of long-term repopulating stem cells without affecting their
engraftment and
multilineage differentiation capacity. Our data further suggest an in vivo
controlled expression
pattern of the transgene, in the absence of exogenous promoters, highlighting
that the
expression is lymphoid specific and limited to immature lymphocytes.
Test corrective donor on hMPB-0034+ cells
Next we designed and tested the corrective AAV6 vector carrying RAG1 coding
sequence. In
particular, the corrective donor included the two homology arms at the 3' and
5' extremities, a
splice acceptor followed by the Kozak sequence, the RAG1 coding sequence and
the BGH
PolyA for a total length of 4.1 Kb (Figure 8 A). RAG1 coding sequence was
codon optimized
replacing more "rare" codons with more frequent ones without changing the
amino acid
sequence, thus enhancing protein translation. We tested the new donor DNA on
hCD34+ cells
obtained from mobilized peripheral blood (MPB) to verify whether the dimension
of the donor
DNA could affect the efficiency of the integration and/or the toxicity
profile.
MPB-CD34+ cells from normal donors (commercially purchased by AlICells
California, US)
were thawed and prestimulated for three days. We adjusted the editing protocol
as follows:
Stem cell factor (SCF) 300 ng/ml, Flt3 ligand (Flt-3L) 300 ng/ml,
Thrombopoietin (TPO) 100
ng/ml, Interleukin 3 (IL-3) 60 ng/ml, StemRegenin1 (SRI, 1 uM) and 16,16-
dimethyl
prostaglandin E2 (dmPGE2, 10uM), UM171 35nM.
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Cas9 was electroporated as in vitro preassembled RNP at two doses (25pm01/well
and
50pm01/well). Since our previous observation suggested that high AAV6 vector
MOI could
impair cell fitness, we considered two low MCI (10k and 2*104).
Impact of the editing procedure was evaluated considering cell growth and cell
phenotype by
flow cytometry. Since the corrective donor does not include any reporter gene,
we assessed
the integration by molecular assays. Four days after editing, cells were
sorted based on the
expression of CD34, CD133, and CD90 to identify and analyze primitive, early
and committed
progenitor subpopulations. Genomic DNA from sorted subpopulations was
extracted, and
targeted integration of the corrective donor was verified by ddPCR assay,
using a set of
primers specific for the on-target integration and for the codon optimized
donor sequence
(Figure 8 B). In accordance with previous observations, the editing protocol
did not affect cell
phenotype based on the expression of CD133 and CD90 (data not shown) and high
on-target
integration frequency was observed in all 0D34 subpopulation. In particular,
in the most
primitive subpopulation a targeting frequency of 45.3% was observed using
50pm01/well Cas9
and 104 MOI of AAV6 vector (Figure 8 C) also showing lower impact on cell
growth as
compared to the higher MOI (Figure 8 D). No differences were noticed between
hCD34+ cells
from MPB or CB both in terms of efficiency and toxicity.
In vivo transplantation of edited hMPB-CD34+ cells from HD and Patient
To assess whether our gene editing procedure may affect engraftment
capability, edited
hMPB-CD34 cells were transplanted into sub-lethally irradiated NSG mice.
Following the
same protocol used in the previous experiment, after 3 days of stimulation,
hMPB-CD34+ cells
were electroporated with 50pm01/well of guide 9 RNP and 15 minutes later
transduced with
corrective AAV6 at MOI 104 Vg/cell. To dampen the previously reported editing-
induced p53
response, which decreases hematopoietic reconstitution by edited HSPCs, we
added to the
electroporation mixture an mRNA encoding for the dominant-negative p53
inhibitor GSE56
(Schiroli G, et al. Cell Stem Cell. 2019;24(4):551-565.e8).
To evaluate in vivo gene correction, we had access to hMPB-CD34+ cells
obtained from a
patient (NIHPID0021) carrying hypomorphic mutations in RAG1 gene. Of note
NIHPID0021 is
an adult patient with CID-G/AI due to missense RAG1 mutations (C1228T; G1520A)
allowing
residual development of B and T cells. The patient presented B cells 23/uL, T
cells 665/uL
(8% naïve), normal NK counts. Of note, the very low B cell counts in the
periphery was also
due to the treatment with anti-CD20 mAb to control severe autoimmune
manifestations.
RAG1 patients received G-CSF/Plerixafor, and CD34+ cells were collected by the
NIH clinical
facility and their purity was verified by flow cytometry (>97% CD34').
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hMPB-CD34+ cells from two independent healthy donors (commercially purchased)
were used
in parallel. The day following the editing, 1x106 of treated or untreated
cells were injected in
sublethally irradiated mice (120 rad) (Figure 9 A). In order to assess the
levels of gene
targeting efficiency after the treatment, few cells were maintained in culture
for four more days.
ddPCR showed a targeted frequency of 86% in patient cells, while 89% and 80%
were
observed in the two healthy donor batches respectively, thus recapitulating
the results
obtained in the previous experiment (Figure 9 B).
Flow cytometric analysis on the peripheral blood was performed 5, 8, 12 weeks
after
transplantation, and mice were sacrificed at 15 weeks.
The analysis of the peripheral blood showed that engraftment of hMPB-CD34+ was
significantly lower than hCB-CD34+. Frequency of hCD45+ cells from HDs
assessed in the
blood was between 4.4% and 8.7% in all time points, and engraftment of the two
batches was
superimposable. Conversely, in CID/AG N IH PI D002 patient the frequency of
hCD45+ cells in
PB was generally lower (between 2.1 and 5.2% at the first two time points) and
decreased at
later time points suggesting exhaustion of the engraftment. Of note, in both
cases (CID/AG
Patient and HD cells) no differences between treated and untreated cells were
noticed in terms
of frequency of hCD45+ cells in PB, confirming that engraftment capability was
not affected by
the editing protocol (Figure 9 C).
Molecular analysis performed by ddPCR assay revealed a targeting frequency of
35.3% in
human cells obtained from peripheral blood of mice receiving gene edited MPB-
CD34+ HD
cells, thus recapitulating previous observations obtained with the reporter
gene and further
confirming that targeting procedure does not affect the engraftment (Figure 9
D). Lower
targeting frequency (9.3%) was obtained in the PB 8 weeks after transplant
with gene edited
MPB patient CD34+ cells (Figure 9 D).
With regard to peripheral blood composition, NSG mice transplanted with
treated HD cells
showed no major skewing in the subpopulation composition and a comparable
frequency of
B, T and myeloid cells was observed in mice receiving treated or untreated
cells, confirming
that multilineage differentiation was not impaired (Figure 9 E). Untreated
patient cells showed
a partial skew in B- and T- cell compartment, when compared to the HD, in line
with the
immune phenotype of patients carrying hypomorphic mutations (Delmonte OM, et
al. Blood.
2020;135(9):610-9). At the last time point, mice receiving untreated patient
cells, B-cell
frequency was 17.2% (HD untreated = 81.9%) and T-cell frequency was 2.3% (HD
untreated
= 9.2%) with a high myeloid cell frequency that was 19.9% (HD untreated =
3.0%). These
observations confirm that despite defects in B- and T-cell development, some
circulating B-
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and T-lymphocytes can be detected. No significant differences were noticed
between mice
receiving untreated or treated patient cells in terms of peripheral blood
immune composition,
even though we observed a slight increase in B-cell frequency in treated
patient cells that was
maintained over time (Figure 9 F).
Mice were sacrificed 17 weeks after the transplant to analyze the engraftment
of edited cells
in bone marrow, thymus and spleen. In the bone marrow and spleen, frequencies
of human
CD45+ cells were higher than those retrieved mice peripheral blood (Figure 8G,
H left panels
and 8C) . NSG mice transplanted with edited MPB CD34 cells from HD showed
13.9% of
hCD45+ in the bone marrow, whilst 23.4% in untreated group (Figure 9 G, left
panel). Similar
engraftment levels were achieved in mice receiving edited RAG1 patient cells
(10.2%), but
lower proportion of hCD45+ cells was found in mice receiving untreated RAG1
patient cells
(6.9%) (Figure 9 G, left panel). hCD45+ cells engraftment was even higher in
the spleen for
both edited and untreated cells of HD and patient. In mice receiving HD cells,
the frequency
of hCD45+ cells was 37.4% and 43.3% in mice with edited or untreated cells,
respectively
(Figure 9 H, left panel), indicating the absence of differences between edited
and not edited
cells. Similarly, the frequency of hCD45+ cells was 24% and 23.7% in mice with
edited or
untreated cells derived from the RAG1-patient, respectively (Figure 9 H, left
panel).
HDR targeting efficiency assessed by ddPCR on DNA samples extracted from bone
marrow
and spleen showed a range from 1.1% to 19.6% in edited cells from the bone
marrow, while
2.1% to 8.5% in the case of patient cells (Figure 9 G, right panel). The
spleen showed the
highest targeting frequency, with a range between 6.1% and 22.2% for mice with
edited HD
cells, and between 11.9% and 14.8% for mice with edited patient cells (Figure
9 H, right
panel).
Overall, these findings confirmed the feasibility of gene editing approach to
target the human
RAG1 locus in HSCs derived from HD and patient with RAG1 mutation. The GE
procedure
did not affect the engraftment capability and the multilineage differentiation
of HSCs.
Discussion
Classical gene-addition based gene therapy strategies rely upon the use of
integrating vectors.
The introduction of new generation vectors, whose improved design confers a
safer integration
profile, alleviated but did not abolish the risk of insertional mutagenesis
caused by vector semi-
random integration into the genome (Doi K, Takeuchi Y. Vol. 65, Uirusu. 2015.
p. 27-36).
Furthermore, the use of ubiquitous promoters dramatically hampers the
physiological
expression of therapeutic transgene whose expression is cell specific or
tightly controlled
during cell cycle.
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RAG1 molecule mediates the site-specific DNA double stranded breaks necessary
for
initiating V(D)J recombination (Oettinger MA, et al. Science. 1990;248(4962)1
517-23). DNA
double strand breaks are per se dangerous lesions that can result in
pathological genome
rearrangements or chromosomal translocations. An important mechanism that
ensures the
fidelity of V(D)J recombination resides in the fine control of RAG1 expression
that is restricted
to specific target cells at specific developmental stages. RAG1 expression
regulation is also
indispensable for the selection of functional, non-self-reactive lymphocyte
through complex
mechanisms of "allelic exclusion" or BCR and TCR receptor editing (Ten Boekel
E, et al.
Immunity. 1998;8(2):199-207).
In the past, several attempts to correct RAG1 deficiency by retrovirus or
lentivirus-mediated
gene transfer have led to variable T and B cell reconstitution with
development of inflammatory
infiltrates and autoimmunity when suboptimal immune reconstitution is achieved
(Pike-
Overzet K, et al. Leukemia. 2011;25(9)1471-83; Pike-Overzet K, et al. Vol.
134, Journal of
Allergy and Clinical Immunology. 2014. p. 242-3; Lagresle-Peyrou C, et al.
Blood.
2006;107(1):63-72; and van Til NP, et al. J Allergy Clin Immunol. 2014;133(4)1
116-23). In
parallel, use of exogenous and ubiquitous promoters may lead to genotoxicity
(Zhang Y, et al.
Advances in Immunology. 2010. p. 93-133; and Papaemmanuil E, et al. Nat Genet.
2014;46(2):116-25).
The development of a gene editing platform represents a strategy to overcome
several issues
raised by conventional gene addition protocol. We have been focusing on HSC-
based genome
editing strategy to correct the broad spectrum of RAG1 deficiencies. To this
end, we designed
a strategy targeting the first RAG1 intron thus replacing the RAG1 coding
sequence entirely
contained in the exon 2. Our strategy has the advantage to cure most of
disease-causing
RAG1 mutations, while conserving the expression of the gene driven by its own
promoter. To
this purpose, we identified the best combination of nuclease reagents and
corrective cDNA
donors in NALM6 and K562 cell lines. Cas9 was electroporated as in vitro
preassembled RNP
in order to ensure a robust and short-term persistence in cells as prolonged
persistence of
Cas9 protein in primary cells could lead to off-target cleavage, potentially
affecting cell
homeostasis and functionality (Kim S, et al. Genome Res. 2014;24(6)1 012-9).
Delivering
Cas9 as preassembled RNP is well tolerated and partially protect the gRNA from
intracellular
degradation thus improving stability and activity of the nuclease (Hendel A,
et al. Nat
Biotechnol. 2015;33(9):985-9). To further improve Cas9 activity profile,
chemically modified
gRNAs were used to enhance the stability, together with High Fidelity Cas9
variant in order to
reduce off-target related toxicity (Vakulskas CA, et al. Nat Med.
2018;24(8):1216-24).
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Prediction analysis of gRNA activity using Cas9 expressing cell line revealed
reliable results
for the guide targeting the intron (guide 9).
Next, we turned to hCB-CD34+ cells. HSPC were prestimulated to favour the
transit through
S/G2 phases when H DR preferably occurs (Genovese P, et al. Nature.
2014;510(7504):235-
40; and Kass EM, Jasin M. Vol. 584, FEBS Letters. 2010. p. 3703-8) resulting
in a moderate
cell expansion while preserving original sternness phenotype considering
expression of CD34,
CD133 and CD90 markers.
Using guide 9 (50pm01/well), Cas9 RNP and AAV6 vector (M01 104) carrying the
PGK_GFP
reporter cassette, we obtained good levels of targeting frequency (40.5%) in
CD34+CD133+CD90+ the most primitive cell subpopulation. Molecular analysis
assessed by
ddPCR analysis showed that the majority of the integration was on target.
Notably, during
Cas9 and AAV6 dosage optimization, we noticed that high MCI of AAV6 had a
strong impact
on cell fitness. In vivo experiments further confirmed in vitro data.
Transplantation of treated
hCB-CD34-' cells in sublethally irradiated NSG mice showed long-term
engraftment both in the
bone marrow and peripheral blood, confirming multi-lineage differentiation
capacity and long-
term engraftment of targeted cells. We also tested SA_GFP cassette in which
GFP expression
is controlled by RAG1 endogenous promoter. In vivo data in NSG mice indicated
a controlled
lymphoid specific expression pattern of the transgene, that was restricted to
immature
lymphocytes in which RAG1 is physiologically expressed. To assess the impact
of the
endogenous RAG1 3'UTR in the donor DNA, we tested different donor constructs
carrying
GFP reporter gene. Analysis of donor AAV6 carrying endogenous RAG1-3'UTR
indicates a
reduction of GFP expression as compared to the level obtained using a donor
with
BGH_PolyA. These data associated with the lack of clinically relevant
mutations in the RAG1
3'UTR so far reported in literature, suggest that this region could be
dispensable in the design
of the corrective donor Finally, SA_GFP_ WPRE did not show advantage in GFP
expression
suggesting that WPRE-mediated expression enhancement could be promoter and
cell line
dependent. Based on this evidence, BGH PolyA sequence that allows the highest
transgene
expression level was cloned in the donor DNA. Furthermore, to further enhance
protein
translation, human RAG1 coding sequence was codon optimized replacing more
"rare"
codons with more frequent ones without changing the final amino acid sequence.
The newly designed donor AAV6 vector (including a SA sequence followed by the
Kozak
sequence, the RAG1 codon optimized followed by BGH_PolyA) was tested also in
hMPB-
CD34+ cells. We observed the same efficiency obtained with the previous
donors, confirming
that our protocol is reproducible using several donors and several HSPC
sources. Moreover,
the multiparametric analysis of HSPC composition in untreated and edited HD
cells showed a
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redistribution of HSPC subtypes in cultured cells as compared to cells
analyzed before the
expansion phase (Figure 10 A). In untreated and edited cells, we observed an
expansion of
hematopoietic stem cells (HSC), multipotent progenitors (MPP) and
multilymphoid progenitors
(M LP) at the expense of common myeloid progenitors (CMP), indicating that
editing protocol
preserves stemness composition (Figure 10 A).
Notably, ddPCR analysis showed more than 80% HDR in total CD34+ cells and 45%
of
targeting frequency was observed in the most primitive (CD133+ CD90+)
subpopulation
subset. In vivo experiments in NSG mice transplanted with treated hMPB-CD34+
cells showed
good level of engraftment and multilineage differentiation capability as those
treated with
unedited cells.
We had access to hMPB-CD34+ cells from a CID-G/AI RAG1 patient carrying
hypomorphic
mutations and presenting with a combined immunodeficiency associated to severe
inflammation and autoimmune signs. We confirmed that the editing procedure did
not affect
the HSPC composition in RAG1-deficient cells (Figure 10 B). Even in this case,
we achieved
86% of targeting frequency as shown by ddPCR analysis. The in vivo transplant
of treated and
untreated cells showed lower engraftment of edited cells in peripheral blood
of NSG mice with
patient cells as compared to HD donor cells. In contrast, comparable
engraftment was
observed in bone marrow and spleen between HD and patient-treated mice,
suggesting that
gene edited patient-derived CD34+ cells preserve the engraftment and multi-
lineage
differentiation capability in vivo comparable analysis of central and
peripheral lymphoid
organs. Severe inflammatory conditions occurring in CID patients and/or
effects of drug
administration (anti-CD20 monoclonal antibody or high doses of corticosteroid)
may influence
the CD34"' cells fitness.
Overall, we have established an efficient and promising genome editing
platform for the
correction of RAG1 deficiency.
Materials and methods
Lenti viral vector production and titration
LVs were produced by transient transfection of 293T cells. 24 hours before
transfection 9x106
cells were plated in a 15 cm dish, 2 hours before transfection Iscove's
Modified Dulbecco's
(IMDM) medium was changed. The required transfer vector (34 pg) was mixed with
9 pg of
VSV-G envelope encoding plasmid, 12.5 pg pMDLg/pRRE, 6.25 pg of REV plasmid
and 15
pg of pADVANTAGE per 15 cm dish. This mixture was added to 293T cells by
calcium
phosphate precipitation. After 12-14 hours the medium was replaced with fresh
complete
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IMDM supplemented with 1mM of sodium butyrate. Collection and filtration of
the supernatant
took place 30 hours after this medium change. Following collection, the LV was
concentrated
500 times by ultracentrifugation (2 hr, 20.000 rpm, 200). A serial dilution
was made of a known
amount of 293T cells infected by the LV. After 3 days genomic DNA (gDNA) of
the different
dilutions was isolated with the DNeasy Blood and Tissue Kit. Vector copy
number (VCN) of
the LV was measured by ddPCR. Titer was calculated by using the following
formula: Titer =
VCN x dilution factor x number of infected 293T cells. p24 HIV protein by
ELISA assay (Abcam
218268) in order to estimate the amount of vector particles and calculate the
relative infectivity
of the vector preparation.
Cas9 inducible Cell lines
NALM6 Cas9 cell line was generated by transducing NALM6 cells with a
lentiviral vector
expressing Cas9 protein under the control of a TET-inducible promoter and with
a vector that
constitutively expresses the TET transactivator (Clackson T. Vol. 7, Gene
Therapy. 2000. p.
120-5). When doxycycline is administered to the culture media, the TET
transactivator can
bind the promoter of the Cas9 and induce its expression in the cells. K562
Cas9 cell line was
generated with the same vector. Doxycycline was administered 24h before
electroporation of
the nuclease. Cell lines were maintained in RPM! 1640 medium supplemented with
10% FBS,
glutamine and penicillin/streptomycin antibiotics (complete medium).
gRNA and RNP assembly
Cas9 protein and custom RNA guides were purchased from Integrated DNA
Technologies
(IDT) and assembled following the manufacturer protocol. To enhance cellular
stability,
chemically modified guide RNAs were used. Briefly crRNA and trRNA were
annealed heating
them at 95 C for 5 minutes and letting them slowly cool down at RT for 10
minutes. Cas9
protein was then incubated for 15 minutes at room temperature with the
annealed guide RNA
fragments, to assemble the ribonucleoprotein (RN F).
Guide sequences are shown in the table below:
Guide 1 TTTTCCGGATCGATGTGA
Guide 2 GACATCTCTGCCGCATCTG
Guide 3 GTGGGTGCTGAATTTCATC
Guide 4 GATTGTGGGCCAAGTAACG
Guide 5 GAAAGTCACTGTTGGTCGA
Guide 6 CAATTTTGAGGTGTTCGTT
Guide 7 GGGTTGAGTTCAACCTAAG
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Guide 8 TTAGCCTCATTGTACTAGC
Guide 9 TCAGATGGCAATGTCGAGA
Guide 10 GCAATTTTGAGGTGTTCGT
Guide 11 ACCAGCCTCGGGATCTCAA
Guide 12 TCAAATCAGTCGGGTTTCC
Guide RAG1K0 CCTTCTCAGCATTCCGA
Guide RAG1K0 AACATCTTCTGTCGCTGACT
When used directly as RNA, the following guide sequences for guides 3, 7, 9
and RAG1K0
may be used:
Guide 3 TGTGGGTGCTGAATTTCATC
Guide 7 GGGGTTGAGTTCAACCTAAG
Guide 9 GTCAGATGGCAATGTCGAGA
Guide RAG1K0 GTACCTTCTCAGCATTCCGA
Mismatch selective endonuclease assay
A T7 endonuclease (T7E1) assay was used to measure indels induced by NHEJ.
Briefly,
gDNA of gene edited cells was extracted and amplified by PCR with primers
flanking the Cas9
RN P target site. The PCR product was denatured, slowly re- annealed and
digested with T7
endonuclease (New England BioLabs) for 1h, 370. T7 nuclease only cut DNA at
sites where
there is a mismatch between the DNA strands, thus between re-annealed wild
type and mutant
alleles. Fragments were separated on LabChip GXII Touch High Resolution DNA
Chip
(PerkinElmer()) and analysed by the provided software. The ratio of the
uncleaved parental
fragment versus cleaved fragments was calculated and it gives a good
estimation of NHEJ
efficiency of the artificial nuclease. Calculation of % NHEJ: (sum cleaved
fragment)/(sum
cleaved fragments + parental fragment) x 100. Primer used for NHEJ assay:
Guides 1, 2, 3 FW CCATAAACACTGTCAGAAGAGG
Guides 1,2, 3 RV GTGTTGCAGATGTCACAGG
Guides 4, 9, 11 FW GAAGTGGTTCATGCAAGAGG
Guides 4, 9, 11 RV GGATGAACATGGAGAAAGCAG
Guides 6, 7, 10 FW GGGGAGAAATGTGTAGGGAAG
Guides 6, 7, 10 RV CTCAAAAACAAAGAAATGGGCG
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Guides 5, 8, 12 FW ATAGGTGGATGGGATGATGG
Guides 5, 8, 12 RV CCTCTTCTGACAGTGTTTATGG
Guides RAG1K0 FW GGAAAATGAATGCCAGGCAG
Guides RAG1K0 RV AGGTCATCATGCTGTACAAATG
Guides RAG1K0 FW TCCATGCTTCCCTACTGAC
Guides RAG1K0 RV CTCCCATTCCATCACAAGAC
10 Off-target analysis
In silico prediction of off-target profile was performed with COSMID (CRISPR
Off-target Sites
with Mismatches, Insertions, and Deletions) (Cradick TJ, et al. Mol Ther -
Nucleic Acids.
2014;3(12):e214) to search genomes for potential CRISPR off-target sites. For
GUIDE-Seq
analysis K562 cells were electroporated with 50 pmol of High Fidelity Cas9
Nuclease V3
guide7 or guide 9 (as RNP) and dsODN to tag the breaks via an end-joining
process consistent
with NHEJ. dsODN integration sites in genomic DNA were precisely mapped at the
nucleotide
level using unbiased amplification and next-generation sequencing (Tsai SO, et
al. Nat
Biotechnol. 2015;33(2)1 87-97). Library construction and GUIDE-Seq sequencing
were
performed by Creative Biogen Biotechnology (NY, USA) using Unique Molecular
Identifier
(UMI) for tracking PCR duplicates. Quality checking and trimming were
performed on the
sequencing reads, using FastQC and Trim_galore, respectively. High quality
reads were
aligned against the human reference genome (GRCh38), using Bowtie2 (Langmead
B,
Salzberg SL. Nat Methods. 2012;9(4):357-9) in the "very-sensitive-local" mode,
in order to
achieve optimal alignments. GUIDE-Seq data analysis was performed employing
the
R/Bioconductor package GUIDE-seq (Zhu LJ, et al. BMC Genomics. 2017;18(1)),
and using
UMI to deduplicate reads.
Donor constructs
The cloning of plasmids was performed using basic molecular biology
techniques. In short,
plasmids were digested using restriction enzymes (New England BioLabs) and
correct
fragments were separated and purified by agarose gel electrophoresis.
Fragments were
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inserted into a dephosphorylated linearized backbone with either Quick Ligase
or T4 Ligase
after purification with QIAquick PCR Purification Kit (QIAGEN). After
ligation, TOP10
chemically competent E. Coll bacteria were transformed and plated on plates
containing
antibiotics. Plasmid DNA was extracted and purified with Wizard Plus SV
Minipreps DNA
Purification System (Promega) and EndoFree Plasmid Maxi Kit (QIAGEN). Colonies
were
screened with control digestions and sequenced. Sequences of vector inserts
with main
features are reported below:
AAV6 vector carrying SA_GFP_BGHPolyA, guide 9:
INSERT
tgagcacacagttattacttggaaattgtgtacagactaagttgaagatgttaggagggaagattgtgggccaagtaac
ggggtgt
atgtgtgtgggtatagggtgggcagctgggatggaaatggggggctgctgctgctgctgcaccctggcctcctgaacta
atgatat
cactcaccagaaactactgttcctgcactgtccaagccaccccaaactagtttgtcaaaatgaatctgtgctgtgtgga
gggaggc
acgcctgtagctctgatgtcagatggcaatgtgaattcctgacctcttctcttcctcccacaggccgccaccatggtga
gcaagggc
gaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccg
gcga
gggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggccc
acc
ctcgtgaccaccctgacctacggcgtgcagtgcttcagtcgctaccccgaccacatgaagcagcacgacttcttcaagt
ccgcca
tgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagtt
cga
gggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctg
g
agtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccg
cca
caacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctg
cc
tgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggag
ttc
gtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaactgtgccttctagttgccagccatctgttg
tttgcccct
cccccgtgccttccttgaccctggaaggtgccactcccactgccctttcctaataaaatgaggaaattgcatcgcattg
tctgagtag
gtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggg
gat
gcggtgggctctatggggatccggaacaggtgtgataatgagagatcttgcgttccaacgagaaactaatgtttctaga
atggca
gtggccggtggggacagggctgagccagcaccaaccactcagcctttgagatcccgaggctggtctactgctgagacct
fttgtt
agaagagaggagatcaagcatttgcaaggfttctgagtgtcaaaatatgaatccaagataactctttcacaatcctaac
ttcatgct
gtctacaggtccatattttagcctgctttctccatgttcatccgaaaagaaagaaaagctaagggtggtggtcatattt
gaaattagc
cagatcttaagttfttctgggggaaatttagaagaaaatatggaaaagtgactatgagcaca
HA Left
tgagcacacagttattacttggaaattgtgtacagactaagttgaagatgttaggagggaagattgtgggccaagtaac
ggggtgt
atgtgtgtgggtatagggtgggcagctgggatggaaatggggggctgctgctgctgctgcaccctggcctcctgaacta
atgatat
cactcaccagaaactactgttcctgcactgtccaagccaccccaaactagtttgtcaaaatgaatctgtgctgtgtgga
gggaggc
acgcctgtagctctgatgtcagatggcaatgt
Splice Acceptor
ctgacctcttctcttcctcccacag
KOZAK
gccgccaccatg
GFP
atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccaca
agt
tcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagct
gcc
cgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgettcagtcgctaccccgaccacatgaagcag
cacg
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acttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatclicttcaaggacgacggcaactacaagac
ccgc
gccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaaca
tc
ctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaagg
tg
aacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcg
ac
ggccccgtgctgctgcctgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatc
ac
atg gtcctg ctgg agttcgtg accg ccg ccgg gatcactctcg gcatg gacgagctgtacaagtaa
PolyA
actgtg ccttctagttg ccag ccatctgttgtttg cccctcccccgtgccttccttgaccctg gaag gtg
ccactcccactgccctttcct
aataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaa
ggggg
aggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgg
HA Right
atggcagtggccggtggggacagggctgagccagcaccaaccactcagcctttgagatcccgaggctggtctactgctg
agac
cfittgttagaagagaggagatcaagcatttgcaaggffictgagtgtcaaaatatgaatccaagataactcfficaca
atcctaactt
catgctgtctacaggtccatattttagcctgcffictccatglicatccgaaaagaaagaaaagctaagggtggtggtc
atatttgaa
attagccagatcttaagtlittctgggggaaatttagaagaaaatatggaaaagtgactatgagcaca
AAV6 vector carrying SA_GFP_WPRE_BGHPolyA, guide 9:
INSERT
tgagcacacagttattacttggaaattgtgtacagactaagttgaagatgttaggagggaagattgtgggccaagtaac
ggggtgt
atgtgtgtgggtatagggtgggcagctg ggatggaaatggggggctgctgctgctgctg
caccctggcctcctgaactaatgatat
cactcaccagaaactactgttcctgcactgtccaagccaccccaaactagtttgtcaaaatgaatctgtgctgtgtgga
gggaggc
acgcctgtagctctgatgtcagatggcaatgtgaattcctgacctcttctcttcctcccacaggccgccaccatggtga
gcaagggc
gaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccg
gcga
gggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggccc
acc
ctcgtgaccaccctgacctacggcgtgcagtgcttcagtcgctaccccgaccacatgaagcagcacgacttcttcaagt
ccgcca
tgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagtt
cga
gggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctg
g
agtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccg
cca
caacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtg ctg
ctg cc
tgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggag
ttc
gtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaactagtactcgacaatcaacctctggattac
aaaatt
tgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatc
atgctattgcttcccg
tatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaa
cgtggcgtggtgtg
cactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttc
cccctccctat
tgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtg
gtgttgt
cggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgt
cccttcggccc
tcaatccagcggaccttccttcccgcgg cctg ctg ccgg ctctgcg gcctcttccgcgtcttcg
ccttcgccctcag acg agtcgg at
ctccctttgggccgcctccccgcctggaatggatcctaaactgtgccttctagttgccagccatctgttglitgcccct
cccccgtgcct
tccttgaccctggaaggtgccactcccactgccctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggt
gtcattctatt
ctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggct
c
tatggtctagaatggcagtggccggtggggacagggctgagccagcaccaaccactcagcctttgagatcccgaggctg
gtct
actgctgagaccttttgttagaagagaggagatcaagcatttgcaaggtttctgagtgtcaaaatatgaatccaagata
actctttca
caatcctaacttcatgctgtctacaggtccatattttagcctgctttctccatgttcatccgaaaagaaagaaaagcta
agggtggtg
gtcatatttgaaattagccagatcttaagttffictgggggaaatttagaagaaaatatggaaaagtgactatgagcac
a
HA Left
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tgagcacacagttattacttggaaattgtgtacagactaagttgaagatgttaggagggaagattgtgggccaagtaac
ggggtgt
atgtgtgtgggtatagggtgggcagctgggatggaaatggggggctgctgctgctgctgcaccctggcctcctgaacta
atgatat
cactcaccagaaactactgttcctgcactgtccaagccaccccaaactagtttgtcaaaatgaatctgtgctgtgtgga
gggaggc
acgcctgtagctctgatgtcagatggcaatgt
Splice Acceptor
ctgacctcttctcttcctcccacag
KOZAK
gccgccaccatg
GFP
Atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccaca
agt
tcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagct
gcc
cgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagtcgctaccccgaccacatgaagcag
cacg
acttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagac
ccgc
gccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaaca
tc
ctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaagg
tg
aacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcg
ac
ggccccgtgctgctgcctgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatc
ac
atggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa
WPRE
aatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggat
acgctgctttaat
gcctligtatcatgctattgcttcccgtatggctlicattlictcctccttgtataaatcctggttgctgtctctttat
gaggagttgtggcccgtt
gtcaggcaacgtggcgtggtgtgcactgtgthgctgacgcaacccccactggttggggcattgccaccacctgtcagct
cctttcc
gggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctc
ggctgttg
ggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattc
tgcgcgggac
gtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctctt
ccgcgtcttc
gccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctg
PolyA
actgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactccca
ctgccctttcct
aataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaa
ggggg
aggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgg
HA Riciht
atggcagtggccggtggggacagggctgagccagcaccaaccactcagcctttgagatcccgaggctggtctactgctg
agac
ctlitgttagaagagaggagatcaagcatttgcaaggtttctgagtgtcaaaatatgaatccaagataactclitcaca
atcctaactt
catgctgtctacaggtccatattttagcctgctttctccatglicatccgaaaagaaagaaaagctaagggtggtggtc
atatttgaa
attagccagatcttaagthttctgggggaaatttagaagaaaatatggaaaagtgactatgagcaca
AAV6 vector carrying SA_GFP_SD, guide 9:
INSERT
tgagcacacagttattacttggaaattgtgtacagactaagttgaagatgttaggagggaagattgtgggccaagtaac
ggggtgt
atgtgtgtgggtatagggtgggcagctgggatggaaatggggggctgctgctgctgctgcaccctggcctcctgaacta
atgatat
cactcaccagaaactactgttcctgcactgtccaagccaccccaaactagtttgtcaaaatgaatctgtgctgtgtgga
gggaggc
acgcctgtagctctgatgtcagatggcaatgtgaattcctgacctcttctcttcctcccacaggccgccaccatggtga
gcaagggc
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gaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccg
gcga
gggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggccc
acc
ctcgtgaccaccctgacctacggcgtgcagtgcttcagtcgctaccccgaccacatgaagcagcacgacttclicaagt
ccgcca
tgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagtt
cga
gggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctg
g
agtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccg
cca
caacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctg
cc
tgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggag
ttc
gtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaactggatccaggtaagttctagaatggcagt
ggcc
ggtggggacagggctgagccagcaccaaccactcagcctttgagatcccgaggctggtctactgctgagaccttttgtt
agaag
agaggagatcaagcatttgcaaggffictgagtgtcaaaatatgaatccaagataactcfficacaatcctaacttcat
gctgtctac
aggtccatattttagcctgclitctccatgttcatccgaaaagaaagaaaagctaagggtggtggtcatatttgaaatt
agccagatc
ttaagttittctgggggaaatttagaagaaaatatggaaaagtgactatgagcaca
HA Left
tgagcacacagttattacttggaaattgtgtacagactaagttgaagatgttaggagggaagattgtgggccaagtaac
ggggtgt
atgtgtgtgggtatagggtgggcagctgggatggaaatggggggctgctgctgctgctgcaccctggcctcctgaacta
atgatat
cactcaccagaaactactgttcctgcactgtccaagccaccccaaactagtttgtcaaaatgaatctgtgctgtgtgga
gggaggc
acgcctgtagctctgatgtcagatggcaatgt
Splice Acceptor
ctgacctcttctcttcctcccacag
KOZAZ
gccgccaccatg
GFP
atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccaca
agt
tcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagct
gcc
cgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagtcgctaccccgaccacatgaagcag
cacg
acttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatclicttcaaggacgacggcaactacaagac
ccgc
gccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaaca
tc
ctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaagg
tg
aacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcg
ac
ggccccgtgctgctgcctgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatc
ac
atggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa
Splice Donor
aggtaagt
HA Right
atggcagtggccggtggggacagggctgagccagcaccaaccactcagcctttgagatcccgaggctggtctactgctg
agac
ctlitgttagaagagaggagatcaagcatttgcaaggtttctgagtgtcaaaatatgaatccaagataactclitcaca
atcctaactt
catgctgtctacaggtccatalittagcctgctlictccatglicatccgaaaagaaagaaaagctaagggtggtggtc
atatttgaa
attagccagatcttaagtttttctgggggaaatttagaagaaaatatggaaaagtgactatgagcaca
AAV6 vector carrying PGK_GFP_BGHPolyA, guide 9:
INSERT
118
CA 03195268 2023-4- 11
TT -17 -Z0Z 99Z5610
6
64669e34e0660eebeebeabee0e600664ealelep460eeae006e0e9aepeeambe66406eeae0666640
3le32e3663e66917
e6BeeolloeBoleaBBBee6406e634e0633ee64664330e0e63666e6344Bee6466e6036
06003e6eeoepee3660e60e66eeollollowooe0606e66e00163e406Bee600061e006004Beeopopoe
6oeo6eobee6leoeooe60000elo6o),6eollo646e3646066oelooe6l000eooe64634000e00066400
0646o
3064obee066o0e0oe0643490446ee6pooe6406ee0660epoe0064e60666e6o666e606600464606e0
4
4689390066099946oe6oBboe66436e6o4664004900064664666booeoublo6ebbe6o666990696466
49
dJe
64200
>ivzoN 017
6693333434343396
339348963396446343334366olbeobb3lboeo6o6e66331336993643483633446466363633364334
464333
6664646246636666464666636626663646636666226660066362062626306363666236204064366
369429336646p666496363096336363693664993696669336369096609690
6343339469439343463
236336906389939646326630
64636636344663644334466ee666346ee4333363343643344363863663 gc
300306664644030e43603634404e66003e04606e06044600460e044044e0e0634346664003e6006
06606e0
6oeee66603466463666p4064366363e666936364446664333623662e30444336364666466663e33
Jelowaad Ned
464293664e6e04649643436946433639
3669666966464643646p4ee64eeee3464446epeee3333e336ee334643e064334464394e99693393
4393 0s
424964984392643343366433090 36p6433
6649964966 6406e06664666e4e46664646464e
46466663ee469903666464496ee6669669446496ee6446ee4396939464644ee9664439449446939
3936864
VH
e38368648438646eeee664e18eee68e6e444eee6666643444446ee4434e6e3
obeueee6mele3466466466emobeeeebeeebeeeebooleopbmoolomobloobelmeleoolbbeoelo46
gz
4364e34pee4334eepeomopee4eBeepowe64e4eeee04646e64344466ee364neoBee34e6e66e6e6ee
6e
0411433962 640643e404664066e60004e6e6maabeopemeemeabembe640666e0e6666466006646
e06642e6eloffiBleepeee6e6oeeoon6o64404e6e6e6leele646466eoee6Boole66664e43406664
6606
le66664061e366e06eleeoe6ee66611e06866666ee06e0e6683666616666466666610pelope0161
6
Bel6e64046pre0634e36lleee66e6leeeeleepoll4303643e33343e33646Bee664033e644334433
64633333 0z
40000644646404e006e00646e404400646peeelbeeoe46406e6oe664e36604040eole6660060060
0e646
opbebbloblool6bleoemebobobeebeboee0000ebeeeobebpooboolbe000eobebpoepeooeeoe64
006436406460000660e6066ow000meoce6e0693024ocooe60060406e3646o6e0660e66e6oleoeco
emboolebeeomeeMbeeoleobboeebeebeobeeoeboobbleoleleplboeeoeoobeoeeoeloeeombe
66406e20e36666poleoee0660e6bebbeeolloe6ole066bee6406e6oleobooee6466pooeoe
60666 g
e6044Bee64668600606000e6eeoepee0660e6oe66ee0440404e0oe0606866800463e4066ee60006
4
9336334689344344386393690
69964839339630339p63469344364683646366394339643339339646343
30e303664000646033640Bee06600e3aeoBloleonBee64000e640Bee0660elooe3064e63666e
60666
26366334646362344628323366322246396366326643626346643349330
646646666332311643626626
066Bee36e61661e03e03630666e0000loppoeBooeoleeBooe6116010001066016e0660160e0606e
66 0 1,
ooloo699064044e0600446466060600364004464030666464694660666046466660669666064660
6666e
266633663623626260360606662o6eop6436636242233664643666426oBooeboo6o6o6236642eo
6e6669306o6e0e66326eo6ol000elbeloeoloT6oeobooBee66oeeeoe646oe663364636636044663
6
woubbeebbbolbeep000boolobpouoboebobb000000bbblbu000moboobouolebb000embobeobo
44630463eollowoe0604046664000e6036366o6e060eee666004466460666404064066063e666e0
606 g
4466643336e366ee3344443363466664466663e33664e3334ee64642e36642
6834648643436846433638
3662666266464643646404226422223464446243222333323362233464020
6433116432432226233234023
leleBleepee640340366pooe06436406436406436666664eee664e6664oBea6664666ele4666464
6464e
46466660ee469e0066646ilebee666e66eilblebeebubeepebeoe4646ileee66443enenbeoeoeob
e64
ZZZSLO/IZOZdJ/Id
tS06LO/ZZOZ OAA
WO 2022/079054
PCT/EP2021/078222
aacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcg
ac
ggccccgtgctgctgcctgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatc
ac
atggtcctgctggagttcgtgaccgccgccg ggatcactctcggcatggacgagctgtacaagtaa
PolyA
actgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactccca
ctgccctttcct
aataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaa
ggggg
aggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgg
HA Riciht
atggcagtggccggtggggacagggctgagccagcaccaaccactcagcctttgagatcccgaggctggtctactgctg
agac
cttttgttagaagagaggagatcaagcatttgcaaggtttctgagtgtcaaaatatgaatccaagataactctttcaca
atcctaactt
catgctgtctacaggtccatattttagcctgctttctccatgttcatccgaaaagaaagaaaagctaagggtggtggtc
atatttgaa
attagccagatcttaagtttttctggg ggaaatttagaagaaaatatggaaaagtgactatgagcaca
AAV6 vector carrying SA_GFP_3'UTR-RAG1, guide 9:
INSERT
tgagcacacagttattacttggaaattgtgtacagactaagttgaagatgttaggagggaagattgtgggccaagtaac
ggggtgt
atgtgtgtgggtatagggtgggcagctgggatggaaatggggggctgctgctgctgctgcaccctggcctcctgaacta
atgatat
cactcaccagaaactactgttcctgcactgtccaagccaccccaaactagtttgtcaaaatgaatctgtgctgtgtgga
gggaggc
acgcctgtagctctgatgtcagatggcaatgtgaattcctgacctcttctcttcctcccacaggccgccaccatggtga
gcaagggc
gaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccg
gcga
gggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggccc
acc
ctcgtgaccaccctgacctacggcgtgcagtgcttcagtcgctaccccgaccacatgaagcagcacgacttcttcaagt
ccgcca
tgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagtt
cga
gggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctg
g
agtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccg
cca
caacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtg ctg
ctg cc
tgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggag
ttc
gtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaactggatccgtagggcaaccacttatgagtt
ggffitt
gcaattgagtttccctctgggttgcattgagggcttctcctagcaccctttactgctgtgtatggggcttcaccatcca
agaggtggtag
gliggagtaagatgctacagatgctctcaagtcaggaatagaaactgatgagctgattgcttgaggcttttagtgagtt
ccgaaaa
gcaacaggaaaaatcagttatctg
aaagctcagtaactcagaacaggagtaactgcaggggaccagagatgagcaaag atc
tgtgtgtgttggggagctgtcatgtaaatcaaagccaaggttgtcaaagaacagccagtgaggccaggaaagaaattgg
tcttgt
ggttttcatttttttcccccttgattgattatattttgtattgagatatgataagtgccttctatttcatttttgaata
attcttcatttttataattttac
atatcttggcttgctatataagattcaaaagagcttlitaaattffictaataatatcttacatttgtacagcatgatg
acctttacaaagtg
ctctcaatgcatttacccattcgttatataaatatgttacatcaggacaactttgagaaaatcagtccttlittatgli
taaattatgtatcta
ttgtaaccttcagagtttaggaggtcatctgctgtcatggatttttcaataatgaatttagaatacacctgttagctac
agttagttattaa
atcttctgataatatatgtttacttagctatcagaagccaagtatgattclitatttttactffitcatttcaagaaat
ttagagtttccaaattta
gagcttctgcatacagtcttaaagccacagaggcttgtaaaaatataggttagcttgatgtctaaaaatatatlicatg
tcttactgaa
acattttgccagactttctccaaatgaaacctgaatcaatlittctaaatctagglitcatagagtcctctcctctgca
atgtgttattctlict
ataatgatcagtttactttcagtggattcagaattgtgtagcaggataaccttgtatttttccatccgctaagtttaga
tggagtccaaac
gcagtacagcagaagagttaacatttacacagtgctttttaccactgtggaatgttttcacactcatttttccttacaa
caattctgagg
agtaggtgttgttattatctccatttgatgggggtttaaatgatttgctcaaagtcatttaggggtaataaatacttgg
cttggaaatttaa
cacagtccttttgtctccaaag cccttcttctttccaccacaaattaatcactatgtttataag gtagtatcag
aatttttttagg attcaca
actaatcactatagcacatgaccttgggattacatttttatggggcaggggtaagcaagtttttaaatcatttgtgtgc
tctggctcttttg
atagaagaaagcaacacaaaagctccaaagggccccctaaccctcttgtggctccagttatttggaaactatgatctgc
atcctta
ggaatctgggatttgccagttgctggcaatgtagagcaggcatggaattttatatgctagtgagtcataatgatatgtt
agtgttaatta
glittliclicclitgalittattggccataattgctactclicatacacagtatatcaaag a g cttg
ataatttag ttg tca aa ag tgcatog
gcgacattatctttaattgtatgtatttggtgcttcttcagggattgaactcagtatctttcattaaaaaacacag
cagttttccttgcttttta
120
CA 03195268 2023-4- 11
WO 2022/079054
PCT/EP2021/078222
tatgcagaatatcaaagtcatttctaatttagttgtcaaaaacatatacatattttaacattagtlittttgaaaactc
ttgglittgffittttgg
aaatgagtgggccactaagccacactttcccttcatcctgcttaatccttccagcatgtctctgcactaataaacagct
aaattcacat
aatcatcctatttactgaagcatggtcatgctgglitatagattlittacccatlictactclitlictctattggtgg
cactgtaaatactttcc
agtattaaattatccttttctaacactgtaggaactattttg aatg catgtg actaagagcatgatttatag
cacaacctttccaataatc
ccttaatcagatcacattttgataaaccctgggaacatctggctgcaggaatttcaatatgtagaaacgctgcctatgg
tffittgccct
tactgttgagactgcaatatcctagaccctagttttatactagagttttatttttagcaatgcctattgcaagtgcaat
tatatactccagg
gaaattcaccacactgaatcg ag catttgtgtgtgtatgtgtgaagtatatactg gg
acttcagaagtgcaatgtatttttctcctgtg a
aacctgaatctacaagttttcctgccaagccactcaggtgcattgcagggaccagtgataatggctgatgaaaattgat
gattggtc
agtgaggtcaaaaggagccttgggattaataaacatgcactgagaagcaagaggag
gagaaaaagatgtctttttcttccaggt
gaactggaatttagttttgcctcagatttttttcccacaagatacagaagaagataaagatttttttggttg
agagtgtgggtcttgcatt
acatcaaacagagttcaaattccacacagataagaggcaggatatataagcgccagtggtagttgggaggaataaacca
ttatt
tggatgcaggtggffittgattgcaaatatgtgtgtgtcttcagtgattgtatgacagatgatgtattcttttgatgtt
aaaagattttaagta
agagtag atacattgtacccatttta cattttcttattttaactacagtaatctacataaatatacctcag
aaatcatttttg gtgattatttttt
gttttgtagaattgcacttcagtttattttcttacaaataaccttacattttgtttaatggcttccaagagccttittt
ttttttgtatttcagag aa
aattcaggtaccaggatgcaatggatttatttgattcaggggacctgtgfficcatgtcaaatgttlicaaataaaatg
aaatatgagtt
tcaata cffittatattttaatatttccattcattaatattatggttattgtcagcaattttatgtttg
aatatttgaaataaaagtttaag atttg a
aaatggtatgtattataatttctattcaaatattaataataatattgagtgcagcatttctagaatggcagtggccggt
ggggacaggg
ctgagccagcaccaaccactcagcctttgagatcccgaggctggtctactgctgagaccttttgttagaagagaggaga
tcaagc
atttgcaag gtttctgagtgtcaaaatatg aatccaag
ataactctttcacaatcctaacttcatgctgtctacaggtccatattttag cc
tgctlictccatglicatccgaaaagaaagaaaagctaagggtggtggtcatatttgaaattagccagatcttaagffi
ttctggggga
aatttagaagaaaatatggaaaagtgactatgagcaca
HA Left
tgagcacacagttattacttggaaattgtgtacagactaagttgaagatgttaggagggaagattgtgggccaagtaac
ggggtgt
atgtgtgtgggtatagggtgggcagctgggatggaaatggggggctgctgctgctgctgcaccctggcctcctgaacta
atgatat
cactcaccagaaactactgttcctgcactgtccaagccaccccaaactagtttgtcaaaatgaatctgtgctgtgtgga
gggaggc
acgcctgtagctctgatgtcagatggcaatgt
Splice Acceptor
ctgacctcttctcttcctcccacag
KOZAK
gccgccaccatg
G FP
atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccaca
agt
tcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagct
gcc
cgtg ccctg gcccaccctcgtg accaccctgacctacgg cgtgcagtg cttcagtcgctaccccg
accacatgaag cagca cg
acttclicaagtccgccatgcccgaaggctacgtccaggagcgcaccatclicttcaaggacgacggcaactacaagac
ccgc
gccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaagg
aggacggcaacatc
ctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaagg
tg
aacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcg
ac
ggccccgtgctgctgcctgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcg
cgatcac
atg gtcctg ctgg agttcgtg accg ccg ccgg gatcactctcg gcatg gacgagctgtacaagtaa
3' U T R
gtagg gcaaccacttatg agttg gtttttg caattg agtttccctctg ggttg cattg ag gg
cttctcctag caccctttactg ctgtgtat
ggggcttcaccatccaagaggtggtaggliggagtaagatgctacagatgctctcaagtcaggaatagaaactgatgag
ctgatt
gcttgaggctlitagtgagttccgaaaagcaacaggaaaaatcagttatctgaaagctcagtaactcagaacaggagta
actgc
aggggaccagagatgagcaaagatctgtgtgtgttggggagctgtcatgtaaatcaaagccaaggligtcaaagaacag
ccag
tgaggccaggaaagaaattggtcttgtgglittcattlitttcccccttgattgattatattttgtattgagatatgat
aagtgccttctatttca
121
CA 03195268 2023-4- 11
WO 2022/079054
PCT/EP2021/078222
thttgaataattcttcatifitataatlitacatatcttg gcttgctatataag attcaaaa gag
cifittaaatttttctaataatatcttacattt
gtacagcatgatgacctttacaaagtgctctcaatgcatttacccattcgttatataaatatgttacatcaggacaact
ttgagaaaat
cagtcclitlitatglitaaattatgtatctattgtaacclicagaglitaggaggtcatctgctgtcatggatlittc
aataatgaatttagaa
tacacctgttagctacagttagttattaaatcttctg ataatatatgtttacttag ctatcag
aagccaagtatgattctttatttttactttttc
atttcaagaaatttagagtttccaaatttagagcttctgcatacagtcttaaagccacagaggcttgtaaaaatatagg
ttagcttgat
gtctaaaaatatatttcatgtcttactgaaacattttgccagactttctccaaatgaaacctgaatcaatttttctaaa
tctaggthcatag
agtcctctcctctg caatgtgttattctttctataatgatcagtttactttcagtg gattcagaattg tgtag
caggataaccttgtatttttcc
atccgctaagtttagatggagtccaaacgcagtacagcagaagagttaacatttacacagtgctttttaccactgtgga
atgttttca
cactcattthccttacaacaattctgaggagtaggtgttgttattatctccatttgatgggggtttaaatgatttgctc
aaagtcatttagg
ggtaataaatacttggcttggaaatttaacacagtccffitgtctccaaagcccttcttcthccaccacaaattaatca
ctatgtttataa
ggtagtatcagaattthttaggattcacaactaatcactatagcacatgaccttgggattacattrnatggggcagggg
taagcaag
tttttaaatcatttgtgtgctctggctcttttgatagaagaaagcaacacaaaagctccaaagggccccctaaccctct
tgtggctcca
gttatttggaaactatgatctgcatccttaggaatctgggatttgccagttgctg
gcaatgtagagcaggcatggaattttatatgctag
tgagtcataatgatatgttagtgttaattagthtttcttcctttgattttattggccataattgctactcttcatacac
agtatatcaaagagct
tgataatttagttgtcaaaagtg catcg gcg acattatctttaattgtatgtatttg gtgcttcttcag gg
attgaactcagtatctttcatta
aaaaacacagcagttttccttgctttttatatg cag
aatatcaaagtcatttctaatttagttgtcaaaaacatatacatattttaacatta
gthttttgaaaactcttggttttgtttttttggaaatgagtgggccactaagccacactttcccttcatcctgcttaat
ccttccagcatgtct
ctgcactaataaacagctaaattcacataatcatcctatttactgaagcatggtcatgctggthatagattttttaccc
atttctactctttt
tctctattggtggcactgtaaatactttccagtattaaattatccttttctaacactgtaggaactattttgaatgcat
gtgactaagagca
tgatttatagcacaacclitccaataatcccttaatcagatcacattttgataaaccctgg
gaacatctggctgcaggaatttcaatat
gtagaaacgctgcctatg gttttttg cccttactgttg ag actg caatatcctagaccctagttttatactag
agttttatttttag caatg c
ctattgcaagtgcaattatatactccagggaaattcaccacactgaatcgagcatttgtgtgtgtatgtgtgaagtata
tactggg act
tcag aagtgcaatgtatttttctcctgtgaaacctg
aatctacaagffitcctgccaagccactcaggtgcattgcagggaccagtg a
taatggctgatgaaaattgatgattggtcagtgaggtcaaaaggagccttgggattaataaacatgcactgagaag
caagagga
ggagaaaaagatgtctttttcttccaggtgaactggaatttagttttgcctcagatthtttcccacaagatacagaaga
agataaaga
thttttggttgagagtgtgggtcttgcattacatcaaacagagttcaaattccacacagataagaggcag
gatatataagcgccagt
ggtagttgggaggaataaaccattatttggatgcaggtggthttgattgcaaatatgtgtgtgtcttcagtgattgtat
gacagatgat
gtattcttttgatgttaaaagatthaagtaagagtagatacattgtacccattttacattttcttattttaactacagt
aatctacataaatat
acctcagaaatcatttttggtgattatthttgthtgtagaattgcacttcagthattttcttacaaataaccttacatt
ttgthaatggcttcc
aagagcctthttthtttgtatttcagagaaaattcaggtaccaggatgcaatggatttatttg
attcaggggacctgtgtttccatgtcaa
atgthtcaaataaaatgaaatatgagthcaatacthttatattttaatatttccattcattaatattatggttattgtc
agcaatthatgtttg
aataffigaaataaaaglitaagatttgaaaatggtatgtattataatlictattcaaatattaataataatattgagt
gcagcatt
HA Riciht
atggcagtggccggtggggacag
ggctgagccagcaccaaccactcagcctitgagatcccgaggctggtctactgctgagac
ctthgttagaagagaggagatcaagcatttgcaaggthctgagtgtcaaaatatgaatccaagataactcthcacaatc
ctaactt
catgctgtctacaggtccatattttagcctgctttctccatgttcatccgaaaagaaagaaaagctaagggtggtggtc
atatttgaa
attagccagatcttaagthttctggg ggaaatttagaagaaaatatggaaaagtgactatgagcaca
AAV6 vector carrying SA_RAG1-CDS_BGHPolyA, guide 9:
INSERT
tgagcacacagttattacttggaaattgtgtacagactaagttgaagatgttaggagggaagattgtgggccaagtaac
ggggtgt
atgtgtgtgggtatagggtgggcagctg ggatggaaatggggggctgctgctgctgctg
caccctggcctcctgaactaatgatat
cactcaccagaaactactgttcctg
cactgtccaagccaccccaaactagthgtcaaaatgaatctgtgctgtgtggagggaggc
acgcctgtagctctgatgtcag
atggcaatgtgaattcctgacctcttctcttcctcccacaggccgccaccatggccgcctccttcc
cacctacccttggattgtcctccgcccctgacgaaattcaacatccccacatcaaattctcggagtggaagttcaagct
ctttcgcgt
gcgctcgttcgaaaagacccccgaggaagcccaaaaggagaagaaagactcattcgaaggaaaacccagcctcgaacag
tccccggccgtcctggacaaggccgacgggcagaagcctgtgccgacccagccgctgctgaaagcgcacccgaaattct
cca
agaagthcacgataacgagaaggcccggggaaaggccatccaccaagcaaaccttagacacctgtgccgcatctgtggg
a
actcattcagagccgacgaacataaccggagataccctgtgcatggccctgtcgacggaaagaccctggggctcctgag
aaa
122
CA 03195268 2023-4- 11
TT -17 -Z0Z 99Z5610
CZ I,
SCIO- OVH
612332 36336
>1 VZON
6e0e00040344040440400e6p
VS gi7
161223661262o1612613136216133632
366e666e6616161361613Tee6Teeeeol6m6epeee0000eoobeeool6peo6pop6pepeee6e3oeopeo
lelebleeloeebpopob6pooeob061061061360666666wee66le66blobeo666466bele46664646464
e
1616666oembee3366616uebeebbbebbeublebeebubeepebeoelblbueeebblpepepbeoeoeobe61
431 VH 017
2323626121326162222661.212222622621112226666613114162211312623
o6e44eee6444e4e31661661666eep6eeee6eee6eeee6004eo446400p44364006e4444me33166e3e
4o461
361231peepoleeoeollppe21262233122612122223161626pm66223611123622312626626262262
1
46m30ebeb4060e046606be6300426e64ll00beo4Oeo0eeooeo6eoo6e643666eoe66664660066l6e
obbleebeplbbleppbbblbboblebbbblobleobbeobeleeoebeebbbpebbebbbbbeeobeoebbeobbb
gc
6166661666666lope4o44eo46166e16261316peo6o4eo6peee66e64eeee4ee4004443o464oe0004
oeoo616
beebbpooebnoolpoblb00000p0006m6n6pleaabeao6n6eplpoblblobeppreebblebopebbeao6
e6e661136e4eBee644ee6664p33e61663403343o66e3333eeBie33e3ll66633p3eBee3433363ee4
e
32363e261231162e6eoopoelBeeoopoele46436613e312362e6136161e6ee66leee63e13616ee33
16e
0063006022612622262111660363011641622322066631626022366626331336661336666312334
661 pc
e6o6oceboTe4426260336463eopboppebeeoeoolloeloceooeTTebeeebbeeboeTebeombeeoTTbee
o
oeboT6p6p6262363062223362322mobeombeolbA363T626633361626622336poobTbop626
636643464o36226426226400m64o3266;eo4oee6363643636226423263226226362333342643226
464
346boboebolbboeeebbeepebleblobeebbelobolpeeebboeeblebboblelle000beebpoeeblebeeb
eeoboopoeoeeeoeblpooep6be3664636eeebeeebee6beeoopo6oeeb000eebeeoe1616ee666 gz
604e
6e64136eoono4e6eeoepn6e6006006oeeo663),eoe6364eoopoo6oe634eo6epo64600eeefte
04p006eee36e04616e666ee616e6e0e6660440ee6ee66163o4ee6oeme4eomee4o4o6o664o166e64
e4
363ee66400ee6e66363e3o3166epeole6opeooll6161plee6eoe316366e66p66333e3363e636131
3
peo6pleoep463op66eopobee66po666e66462e6o6o646op6eeee6oe6w446600ee6666ao4p4e
ollbeellipe6636nne1663666136e6613612613eeboopolbeeble3366e66626e663631e61313363
1613 OZ
44e00600964000eee6oeo4e6o6e6e6oe663661461e64006464o6006eeo64o64o4o6e6eo4oee6006
eeoo
6626226304222343226233opbeleobobuemeolebee61231233231popuo6361633662222663
3646616p32663344663236226266346463263666422664263646342266223466466466323433366
63
eeopoepeboebbplebeeoeolebebleebbbebbpole3ebbebbebbleoebble61333663161661o63633
pe6oelebeop6632eeoo6oleooele6616pooepe6Te661613133133161336636e6136661e6pepeo66
616 91.
oe633e33463p346463eeeee6113336336e3664ee6omeoleooele6663361364p466eeee6e363ee66
3
Tpoo6aeoopeoo6eopple6e366oe6600eoleao66ee616pe66eoeT6Te6eeoemelee000p6Teoppo
neaeoee3163633)reo3631346}6161366336eoope66631e66eee3666e36leoleo366e6613ee6le6
136e
eoe6eoeme6oee66olo666o64o6o664o64444464000e64e0646463046ee6464e6e66e666e6ee66ee
oe
63360110366ee046eeo64o6ee6p6e666331366e3e36ee6eo33666336o4oe6136313136p4eo6e366
3 0 1,
o3o66o466o66beeoeelleoeo61644pleee6eeeool6e66eeoeoobeo6eoleoeooeooeeoe4Oeeee66p
o
3161622662612236162662223633316122231661261333132261361633161131133162231663333
1626613
Te6pepoombp000e466306poll000ftepolobbbleolbbeebpobp6o6po4e36464636006p44646oeo
beeobpeepebebblboomeboobopueleobebobplebembpbemeombeeblbolpeobebboompebb
466366p6pbeemep;6poeooweeeobeofteeo3634ebeebee6;e6464ebbeep],eop4e6633366e33 g
o 636326o
bee3236e33633366e36631366233e66;361633ebeelp6eebeeobebpbe3613Tee6336e3
6133316ee66e6eeope6636366op6peoem6pleoe636133163opeoeoeoole36616e66Tepe616oee
0606000443e4616ee6161333636e0406e0446ee6633e064e44eo6e661364oeeaeoo6pp6e600eemo
eoo
Teobeoebblblebeabbeeblboeboleebeon6166eeeobolebpoe663366poleoebobbbebeebebbee6
ZZZSLO/IZOZdJ/Id tS06LO/ZZOZ OAA
WO 2022/079054
PCT/EP2021/078222
ctgacctcttctcttcctcccacaggtacctcagccagcatggccgcctccttcccacctacccttggattgtcctccg
cccctgacg
aaattcaacatccccacatcaaattctcggagtggaagttcaagctctttcgcgtgcgctcgttcgaaaagacccccga
ggaagc
ccaaaaggagaagaaagactcattcgaaggaaaacccagcctcgaacagtccccggccgtcctggacaaggccgacggg
cagaagcctgtgccgacccagccgctgctgaaagcgcacccgaaattctccaagaagtttcacgataacgagaaggccc
gg
ggaaaggccatccaccaagcaaaccttagacacctgtgccgcatctgtgggaactcattcagagccgacgaacataacc
gga
gataccctgtgcatggccctgtcgacggaaagaccctggggctcctgagaaagaaggagaagagggcgacatcctggcc
gg
acctgatcgcaaaggtgttcagaatcgacgtgaaggcagatgtggacagcatccacccaaccgagttctgccacaactg
ctgg
agcattatgcaccggaagttcagctcagcgccctgtgaagtgtacttcccgcgcaacgtgactatggagtggcatccac
acactc
cgtcctgcgacatctgtaacactgctcggcgcggactcaagaggaagtccctgcagccgaatctgcagctgagcaagaa
gctt
aagaccgtgctggaccaggctcggcaggcccgccagcacaagcgacgcgcccaggcccggatctcatctaaggatgtga
tg
aagaagatcgccaattgcagcaaaatccacctgtctaccaagctgctggcggtggacttcccggagcacttcgtgaagt
ccatc
agctgtcagatctgcgagcatattctcgccgaccccgtggagactaattgcaagcacgtgttctgccgcgtgtgcatcc
tgcgctgc
ctgaaggtcatgggctcctattgcccttcctgccggtacccctgtttccctactgatctggagtccccggtcaagtcct
tcttgtccgtgc
tgaactccctgatggtcaaatgtcccgcaaaggagtgcaatgaggaagtgtccctggaaaagtacaaccaccacatcag
cag
ccacaaggagtccaaagaaatctligtgcacattaacaagggcggtcggccccggcagcatctgctctcgctgactcgc
cgggc
ccagaagcacaggctccgggagctgaagctgcaagtcaaggccttcgccgacaaggaagagggaggagatgtgaagtcc
g
tgtgcatgaccctgffittgctggcgctgcgggctcggaacgaacacagacaagctgatgaactggaggccatcatgca
gggca
aaggatcgggactccagccggctgtgtgtctcgccatccgcgtcaacacattcctctcatgctcccaataccacaagat
gtacag
gactgtgaaggccatcaccggacggcagatctttcagccactccacgcccttcggaacgcagaaaaggtcttgctgccg
ggat
accatcatttcgaatggcagccgcccttgaaaaacgtgtcctcgtccaccgacgtgggcattattgatgggctgagcgg
cctgtcc
tcctctgtggatgactaccctgtggataccatcgccaaacggttcagatacgattccgcgctggtgtcggccctgatgg
acatgga
ggaggacatcctggagggaatgagatcacaagatctggacgactacctcaacgggcccttcacggtggtggtcaaggaa
tcgt
gcgatggaatgggcgacgtgtcggagaagcacggttccggacctgtggtgccggaaaaggccgtgcgcttctccttcac
catca
tgaagatcaccattgcgcatagctcccagaacgtcaaagtgttcgaagaggccaagccgaactcagagctctgctgcaa
gccg
ctgtgcctgatgttggcggacgagagcgatcacgaaaccctgaccgccattctgtcgcctctgatcgcggagagggagg
ccatg
aagtcctccgaactgatgctggagctgggcggtattttgcggacttttaagttcatcttccggggaaccggttatgacg
aaaagctc
gtgcgcgaagtggagggcctggaagcctcaggctccgtctacatctgcactctctgcgacgccacccggctggaggcgt
caca
gaatcttgtgttccactcgatcactaggtcccacgcggagaacctggaacgctatgaggtctggcgctctaacccatac
cacgaa
tccgtggaagaacttcgggacagagtgaagggagtgtcagcaaagcctttcattgaaaccgtgcctagcatcgacgccc
tccat
tgcgacatcggcaacgccgccgagttctacaagatcttccagcttgagatcggggaagtgtacaagaacccgaacgcct
ccaa
ggaagaaagaaagcggtggcaggctacccttgacaaacacctccgcaagaagatgaaectgaagcccattatgeggatg
aa
cggaaacttcgctaggaagctgatgactaaggaaacggtcgacgcggtctgtgaactgatccccagcgaagaacgacat
gaa
gcgctgcgcgaactcatggacctgtacctgaagatgaagcctgtctggcggagctcgtgccctgccaaggagtgcccgg
agtc
gctgtgtcagtacagctttaacagccaaaggttcgcagagctgctgtcgaccaagttcaagtacagatacgaaggaaag
attac
caactacttccacaagactctcgctcacgtgcccgagattatcgaacgcgatggttccatcggggcctgggcctccgag
ggcaa
cgagtcgggcaacaagttgttccgccggtttagaaagatgaacgcccgccagtccaagtgctacgaaatggaagatgtg
ctga
agcatcactggctgtatacctccaagtacctccagaagttcatgaacgcacataacgccctcaagacctccgggttcac
catgaa
cccccaggcctccctcggtgaccctctgggaattgaagatagettggagagccaggactcgatggaattctagctgtgc
cttctag
ttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaa
taaaatgagga
aattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgg
gaaga
caatagcaggcatgctggggatgcggtgggctctatggatccggaacaggtgtgataatgagagatcttgcgttccaac
gagaa
actaatgtt
PoIvA
gctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactccca
ctgtcctttcct
aataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaa
ggggg
aggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgg
HA Right
atggcagtggccggtggggacagggctgagccagcaccaaccactcagcctttgagatcccgaggctggtctactgctg
agac
cttttgttagaagagaggagatcaagcatttgcaaggtttctgagtgtcaaaatatgaatccaagataactctttcaca
atcctaactt
catgctgtctacaggtccatattttagcctgctttctccatglicatccgaaaagaaagaaaagctaagggtggtggtc
atatttgaa
attagccagatcttaagttffictgggggaaatttagaagaaaatatggaaaagtgactatgagcaca
124
CA 03195268 2023-4- 11
WO 2022/079054
PCT/EP2021/078222
AAV6 vector carrying PGK_GFP_BGHPolyA, guide 3:
INSERT
gcaaagatgaatcaaagattctgtccttaaagaccttaaggtttttgtggaaggaaataaaactttacatgtatatatt
taagcactta
tatgtgtgtaacaggtataagtaaccataaacactgtcagaagaggaaataactctatgatcagcacctaacatgatat
attaagg
tagaagatttaatacatatcttttggaatacatgaataaataattgaatgtatttatttttattatttataagatacat
cagtgggatattgat
attggtcttaatatgacttglittcattgttctcaggtacctcagccagcatggcagcctctttcccacccaccttggg
actcagttctgcc
ccagataccggtatggccacggggttggggttgcgccttttccaaggcagccctgggtttgcgcagggacgcggctgct
ctgggc
gtggttccgggaaacgcagcggcgccgaccctgggtctcgcacattcttcacgtccgttcgcagcgtcacccggatctt
cgccgct
acccttgtgggccccccggcgacgcttcctgctccgcccctaagtcgggaaggttecttgcggttcgcggcgtgccgga
cgtgac
aaacggaagccgcacgtctcactagtaccctcgcagacggacagcgccagggagcaatggcagcgcgccgaccgcgatg
ggctgtggccaatagcggctgctcagcagggcgcgccgagagcagcggccgggaaggggcggtgcgggaggcggggtgt
ggggcggtagtgtgggccctgttcctgcccgcgcggtgttccgcattctgcaagcctccggagcgcacgtcggcagtcg
gctccc
tcgttgaccgaatcaccgacctctctccccagggccgccaccatggtgagcaagggcgaggagctgttcaccggggtgg
tgcc
catcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctac
gg
caagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctac
ggcgt
gcagtgcttcagtcgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccag
gagc
gcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccg
cat
cgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaac
g
tctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgt
gc
agctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcctgacaaccactacctgagcac
cca
gtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcact
ctcg
gcatggacgagctgtacaagtaaactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttcctt
gaccctggaa
ggtgccactcccactgccctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctgg
ggggtggggt
ggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggggatccgg
a
acaggtgtgataatgagagatcttgcgttccaacgagaaactaatgtttctagaaattcagcacccacatattaaattt
tcagaatg
gaaatttaagctgttccgggtgagatcctttgaaaagacacctgaagaagctcaaaaggaaaagaaggattcctttgag
ggga
aaccctctctggagcaatctccagcagtcctggacaaggctgatggtcagaagccagtcccaactcagccattgttaaa
agccc
accctaagttttcaaagaaatttcacgacaacgagaaagcaagaggcaaagcgatccatcaagccaaccttcgacatct
ctgc
cgcatctgtgggaattcttttagagctgatgagcacaacaggagatatccagtccatggtcctgtggatggtaaaaccc
taggcctt
ttacgaaagaaggaaaagagagc
HA Left
gcaaagatgaatcaaagattctgtccttaaagaccttaaggtttttgtggaaggaaataaaactttacatgtatatatt
taagcactta
tatgtgtgtaacaggtataagtaaccataaacactgtcagaagaggaaataactctatgatcagcacctaacatgatat
attaagg
tagaagatttaatacatatcttttggaatacatgaataaataattgaatgtatttatttttattatttataagatacat
cagtgggatattgat
attggtcttaatatgacttgttttcattgttctcaggtacctcagccagcatggcagcctctttcccacccaccttggg
actcagttctgcc
ccagat
PGK promoter
ccacggggttggggttgcgccttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccgg
gaaacg
cagcggcgccgaccctgggtctcgcacattcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttgtg
ggccccc
cggcgacgcttcctgctccgcccctaagtcgggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagc
cgca
cgtctcactagtaccctcgcagacggacagcgccagggagcaatggcagcgcgccgaccgcgatgggctgtggccaata
gc
ggctgctcagcagggcgcgccgagagcagcggccgggaaggggcggtgcgggaggcggggtgtggggcggtagtgtggg
ccctgttcctg cccg cgcg gtg ttccg cattctg caagcctccgg ag cgcacgtcgg cag tcgg
ctccctcgttgaccgaatcacc
gacctctctccccagg
KOZAK
ccatg
125
CA 03195268 2023-4- 11
TT -17 -Z0Z 99Z5610
9Z1-
beebbplleoebbeboeeeebbebleeoebbponoebbeeoebbeeolelleeeebpbpleboemeleoeobbbpo
opoboeeo0bolebee6646obboopleeeMboopebolp646eboweeebeeolpepebbebeeebpbeob
eeblbooebibeeebbooeeooebeeopbpbpoebblbowoobbeeeeebeobebobbobebpolpob000beee
9i7
bebleebbbebooeblboeleeeblbeeeooebpbeboemeAbooeollombeboelbpbpobeoeobee000bp
646bee6eboeemobpoee6emebolloeeooMebbobebow34pbebe000boollobobbbeeoeM6616e
e66e6olpee66p0000eoleooeee66e6o6e6eeeBeooeble66poBope6eobeoeee6666eoo66ppoo
666),B3e3ep33ole3Bo3llo3e6433leBeeBeB3leBeeee66633ee3eBBee633pe333ellmeBee6Bea6
bobbobloneoaboeobpbebebbbpaeoolebemeamooleabeabboeeoebolpoebbobeabeebbobp61 ov
ooe66e6ebeoeebpbeeblbop6peebbebooeobboebblebeeeebbpoleombeeoleollbeeoepllbeb
eebbembeoobebbobboeNeoepbbooboepbboeebeeobebeooebo4p444e6ebeeeoelbeebebpo
bpbeobeobboblboppbeee6p6pooebpoebbeooeooeobeboeboelebebeeolebleppobobebpoo
000bbeeooeoTebebooeoeebTbebebpoTeoebobebpbpoTeooboebooTbpoeebeeooboobbpmbpo
ebooboelbeooebobbolebemobbpbpoeeoebbpoeboeboeboepoeoebbeeobeblobeobpeeeoob ge
4ebbeboobb00eb0440eeo6ebee0440ee00000eb400666pobebpooblleb400eeebb04 00bbwebee
beebebobb000bpbe000bolebpleeeebbpbboebeobebeeobebpebeoobplbpowoobbeeooboeb
616066o6e3o6oeeoleoomeeee66e63116p6eooeeoepoe6e06166p6eooleop6p6eeoe6616oe6o
6e3ee3eB0330ee6430e63666e63le630lloeo3666633pBee3leBleoe0336B3036634ep4e6p6636
paebaobbeeaeboaeobeoe66156peeebeeebebpaeaaepleoaeaaaaelbeebebaeaaepabblbbeb
oebblboleoeeobbollole0000eobbobeboeobeebeelebbebee66466popoolBebeebbpebeoeoolp4
4
obeoeboebblbbeeoobblebeboeeobeoTplebebeeobplepbplebbooeebeebboebemeoelebeebe
6200600 40 000200 000
3332634 36 333332 62262234
eo6eoeo6600e600eoeeo666p6466eeowee6eeo6e0006466eeoeT6e6oe600eoTe64630666406664
bppeemeobboleoebbpobboleobeombeebeeoeboobeobe000lbebboeoole166346beebbobeebe
gz
26 2033066 6 63264 3868 38 3
66 3 o86866o86oeoo866o866W
0-ind¨>19d-6se0-131 6u ytueo .101.38A 12-1!Aglie1
6ebebeeeebbeebeeeboepuo0662403322embblebblb004664200 oz
46eoo4ele6e6BeoeeoeoBe64e6p6e6e4mollee66646p4eoBoo6p4o4eoeBollooeemBeeowoo4e6o6
eeea66ebeea6eeebeboeeaebaeaprleeebeeeallp6eepaaeaoabeeeenbileaabeapeeaaalbeoab
ee6ea4664e6p6beeoe66pa),BeoBeaopleeaBe66pppooeee6666e6BponeBBeeBeeee6Beeee
opbeebeebpaeoebeeeebmoolebeblObboollblobeellleeebbleebeollpeeepeleoe000eobeonee
VH
bbleppbbblbboblebbbbpbleobbeobeleeoebeebbbuebbe
66666e2362326623666646666466666643424344204646624626434644236042364422266264eee
e4ee
4334443336p2333432336466226643332644334433646333334333364446446431
233623364462434433 646p2
VAlod
embeeombpbe6oebbleobboppeolebbbooboobooeblboubebbp6polbble 01,
oeowbobobeebeboee0000ebeeeobebpooboolbe000eobebpoepeooeeoebpobp6p64b0000bb
oebobboTe00000eoeebeobeooepeooeboobopbeoblbobeobboebbebo)goeeoeooboolebeeolpee
646beeowobboee6eebeobeeoebooMeolmep46oeeoeoobeoeeoepeeombebbpbeeoeo6666p
0480880660866866ee0440e604e0666ee6p6e604e0600ee6166p00e0e63666e60446ee6166e6006
a6aaaeBeeoepee366ae6ae66eeallanoleaoea6a6e66eaal6aep66ee6aaa6Teaa6aai6eeapapae
g
6oeo6eobee6leaeooe60000elo6o),6eono616ea646366oepoe6pooemeblbopooe00066poo646o
oobpbeeobbooeooeobpleollbee6pooebpbeeobboepoeooble6obbbebobbbebobboolblbobeol
lbeeoeoobboeemboe6obboe66p6e6o166pole00061661666booeollbpbebbebob6beeobe61661e
clA0
ZZZSLO/IZOZdJ/Id tS06LO/ZZOZ OAA
TT -17 -Z0Z 99Z5610
36433643o
6336o4eopee663663e3o644e4o3o4o33o3444o6o4oe6663344o3p6e346p3e33e33644e366 gg
661166pe000meeo6oe6p6m616peo61616616066163ee366e316p60006616p6e66e6TemopT6p61
466poleeele46poolooloppeoplo664e46333)336ile4364eale464n3364eemo64363ele66464e4
363elmoo
43644blepeello4e46643e64ebeee646444eeeeoellebblolooeeoleeoebooee166633334364e3e
6443oeb
p4le63e61434363e613613361361ele61133e6ppe6oe6p0006Te6336eo33436Teoe6epoe6llpe6o
e61433
363ebooe600666o66o6eeeboOl6eeelo6eoeeebebbi.00bblbleleoleepee6610066011lloopoo6
looe OS
eboobebbbeabbooebonblobebpeeobeeoebeblopoe00000bleppebooeooepoeoebebebeeebbe
beeeeobe4beeoleobebbeoeebbe6644e4636406664oeoemoeoo6664633603464o4obombpeo6oee6
00040 00 00 06040 00000e
0000b000eo66op
wo64beemo6666oe6o604e0e040400404064643633e4e346ee3363ee3ee6636),34
3664e346e63
bbeebbl00000blolloemoeleowobbeoebbloblebebolee33613336lebolobl000bbbobeeoeebee6
16
3e366pel64333e4336e36e6116e666436eeee3436340eee66eeoe63e6po66ee6o4e466o464664ee
ol
aepee6643436e663eeele346eee36e6ee3E6613e6e43464ee334664333eeee6ee64464e64664614
3e343
oiolon6be6e166ee60061660006eeob000e6Teo6166loaeo6o600e66ee6o3o6166e6o16oe6o36oa
eo
1633e3113663136636e6oelon333313oee36o33o63633133e6e6613343363336166663363636e63
366
obbe6646ebb0000lobi.boTboobobeo666121666eeobbbemeooeb000boTolbobboTbooeoobbpoub
b afr
Tbob0006e6bee000bbooeoboobobbTooToo6beebbTebeoeeobeo6oboo664o6b000446636e6446eb
oo
66Teo6ob000bbole6ebooboT16),66o6666636eebolbobe6ebboo6oeooebbp166366166oboobobb
o
e6oe66363166616166eeo66o4eoe6o4o666o46o6o6oeo4004jo4oee6eeo64o6e600eo4666o6e6o4
eoe
oobooebbooTebo4booeoeooboboeoob0000moeboobo4bobooboobopooeobomboobbbe00004boeb
oebob000embolooboblbboe000beeoelbebooeblemeoueeobe33331313133ebooeoleebooebpbol
gc
000p66346eo66346oeo6o6e6600loo6eeo6pileo6o3116466o6o60006pop6poo6664646e4663666
6
1646666366e666361663666beebbboobbobeabe6eboobabob6beabeoloblobbobeleeoo66161366
61e63600e6006o6o6e366Teeo6e666eao6o6eoe66oe6eo6opooeT6epeopT6aeo6o36ee66oeee
3e6163e6633616366o634166364331166ee666o16eep3o363313613311363e63663333336661611
3o3e
loboobollow66000eolbobeobollboolboeopolleoeobolo1666l000eboo6obbobeoboeeebbboop
6616 oc
3666ToTobTo6boboebbbeoboblp,666T000beobbecoompobo64466664466663oTebbeeTbeeeeebe
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____________________________________________________________________ Jelowaid
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WO 2022/079054
PCT/EP2021/078222
HSCs were stained with phycoerythrin cyanine 7 (PECy7) 0D34 (Clone: A0136,
Miltenyi
Biotec), phycoerythrin (PE) CD133 (Miltenyi Biotec) allophycocyanin (APC) CD90
(BD
Biosciences). Cell sorting on CD133/CD90 edited cells was performed using
MoFlo XDP Cell
Sorter (Beckman Coulter).
For mice analysis single-cell suspensions were obtained from bone marrow,
spleen, thymus
and peripheral blood and stained with the following anti-human antibodies:
0D45 (clone
REA757), CD3(clone REA613) (Miltenyi biotech), CD19 (clone SJ25C1), CD13
(clone WM15)
(BD Biosciences). Human and murine Fc blocking was performed before each
staining using
human F-Block and murine CD16/CD32 from BD Pharmingen. Live/Dead Fixable
Yellow
(Thermo Fisher Scientific, Waltham, MA) was added to the antibody mix to
exclude dead cells.
Samples were acquired on a FACSCanto ll (BD) and analyzed with FlowJo software
(TreeStar, Ashland, Ore).
Analysis of HSPC composition of MPB-CD34+ cells was performed according to the
protocol
described in Basso-Ricci L, et al. Cytom Part A. 2017; 91(10):952-65. Briefly,
1.5x105 cells
were labeled with fluorescent antibodies against CD3, CD56, CD14, CD61/41,
CD135, CD34,
CD45RA (Biolegend) and CD33, CD66b, CD38, CD45, CD90, CD10, CD11c, CD19, CD7,
and CD71 (BD Biosciences). All samples were acquired through BD LSR-Fortessa
(BD
Bioscience) cytofluorimeter after Rainbow beads (Spherotech) calibration and
raw data were
collected through DIVA software (BD Biosciences). The data were subsequently
analyzed with
FlowJo software Version 9.3.2 (TreeStar) and the graphical output was
automatically
generated through Prism 6.0c (GraphPad software).
AA V6 production and titration
AAV vectors were produced by transient triple transfection of HEK293 cells by
calcium
phosphate. The following day, the medium was changed with serum-free DMEM and
cells
were harvested 72 hours after transfection. Cells were lysed by three rounds
of freeze-thaw
to release the viral particles and the lysate was incubated with DNAsel and
RNAse I to
eliminate nucleic acids. AAV vector was then purified by two sequential rounds
of Cesium
Cloride (CsCl2) gradient. For each viral preparation, physical titres (genome
copies/mL) were
determined by PCR quantification using TaqMan.
AA V6 gene-editing protocol in cell lines
2x105/ 5x105 cells per well were electroporated (Lonza, SF Cell line 4D
Nucleofector X Kit,
program FF120 for K562 or program DC100 for NALM6) with either plasmids or RN
Ps. Fifteen
130
CA 03195268 2023-4- 11
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PCT/EP2021/078222
minutes after electroporation, cells were infected with AAV6 at different M01:
104; 5x104; 105
Vector Genome/cell, Vg/cell.
CD34+ cells
Human cord blood CD34+ cells (CB CD34+ cells) were obtained from Lonza
(PoieticsTM cat#
20 101). CB 0D34+ cells/ml were stimulated in StemSpan medium supplemented
with
penicillin/streptomycin antibiotics and early-acting cytokines: Stem cell
factor (SCF) 100 ng/ml,
Flt3 ligand (Flts-L) 100 ng/ml, Thrombopoietin (TPO) 20 ng/ml, Interleukin 6
(IL- 6) 20 ng/ml,
StemRegenin1 (SRI) (1 uM) and 16,16-dimethyl prostaglandin E2 (dmPGE2) (10uM),
UM171
50nM. Patient mobilized peripheral blood 0D34+ cells (CB 0D34+ cells) were
kindly provided
by Dr. Luigi Notarangelo (Laboratory of Clinical Immunology and Microbiology,
Division of
Intramural Research, National Institute of Allergy and Infectious Diseases,
National Institutes
of Health, Bethesda, MD, United States). MPB 0D34+ cells/ml were stimulated in
StemSpan
medium supplemented with penicillin/streptomycin antibiotics and early-acting
cytokines:
Stem cell factor (SCF) 300 ng/ml, Flt3 ligand (Flts-L) 300 ng/ml,
Thrombopoietin (TPO) 100
ng/ml, Interleukin 3 (IL- 3) 60 ng/ml, StemRegeninl (SR1) (1 uM) and 16,16-
dimethyl
prostaglandin E2 (dmPGE2) (10uM), UM 171 50nM.
AAV6 gene-editing protocol in CD34+ cells
After 3 days of expansion 2x105 CD34+ cells per condition were electroporated
(Lonza, P3
Primary Cell 4DNucleofector X Kit, CD34+ program) with RNPs, GSE56 mRNA (p53
inhibitor)
was added at a dose of 150pg/m1 when cells were aimed at being transplanted.
15 minutes
after electroporation, CD34+ cells were infected with AAV6 at different M01:
104; 5x104; 105
Vg/cell.
Digital PCR
Digital PCR (ddPCR) was performed to assess targeted integration. In short,
gDNA was
quantified using Nanodrop, and diluted in H20 to reach 5-10 ng per reaction (1-
2ng/u1). It is
possible to increase the gDNA quantity per reaction but it is important to
remain below the
saturation limit of the system. ddPCR master mix was prepared by adding 11 ul
ddPCR
Supermix for Probes (no dUTP; BioRad), 1.1 ul primer mix Primer forward +
Primer reverse
(final concentration 0,9uM) + Probe (final concentration 0,25 uM), 1.1 ul
normalizer primer mix,
4.9 ul H20 per reaction. Finally, 17 ul of ddPCR master mix and 5 ul of
diluted gDNA were
added to each well (we included UT and H20 as negative controls, and mono- or
bi allelic
clone as positive control to validate the system). Droplets were prepared on
the BioRad
AutoDG Automated Droplet Generator and the droplet plate was sealed with foil
using BioRad
131
CA 03195268 2023-4- 11
WO 2022/079054 PCT/EP2021/078222
PX1 PCR Plate Sealer. The sealed plate was placed into BioRad T100 Thermal
Cycler and
we ran the appropriate PCR program. The run was read in BioRad QX200 Droplet
Reader.
Calculation copies per genome: concentration (copies/pi) gene of interest /
concentration
(copies/p1) normalizer gene x 2 Calculation percentage of HDR: copies per
genome x 100.
Optimized PCR program (40 cycles):
= 95 C x 10 min
= 40 x 94 x 30 sec
= 55 x 1 min
= 72 x 2 min
= 98 x 10 min
= 4 hold
Primers and Probes used for ddPCR assay are the following:
PGK_GFP cassette FW CAAGAGGTTGTCTGAAGGAAG
PGK_GFP cassette RV GACGTGAAGAATGTGCGAG
PGK_GFP cassette PROBE FAM CTGCTGCACCCTGGCCTCCTGAACTAA
Corrective CDS FW GTGGAACAGGTGTGATAATGAG
Corrective CDS RV GGAGGACAATCCAAGGGTAG
Corrective CDS PROBE FAM TGCTGCTGCACCCTGGCCTCCTGAA
Mice and transplantation protocol
NOD-scid IL2Rgnull mice (NSG; Charles River) were purchased from Charles River
Laboratories Inc. (Calco, Italy) and were maintained in specific pathogen-free
(SPF)
conditions. Mice were transplanted at 8-10 weeks approximately 6 hours after
sublethal total
body irradiation (120 rad), via intravenous injection of treated HSCPs in
phosphate-buffered
saline. Gentamicin sulfate (Italfarmaco, Milan, Italy) was administered in
drinking water
(8mg/mL) for the first 2 weeks after transplantation to prevent infections.
Mice were followed
until the sacrifice and then euthanized for ex vivo analyses.
Statistical analysis
When normality assumptions were not met, non-parametric statistical tests were
performed.
Kruskal-Wallis test with multiple comparison post-test was performed when
comparing more
groups. When normality assumptions were met, two-way analysis of variance
(ANOVA) was
used. For repeated measures over time, two-way ANOVA with Bonferroni's
multiple
comparison post-test was utilized. Values are expressed as Mean SD.
132
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PCT/EP2021/078222
EXAMPLE 2
Results and discussion
Corrective donor screening
To further explore the role of the 3'UTR and the selection strategy, further
corrective donor
sequences numbered 5-8 below were designed and compared with the sequences
numbered
1-4 below (Figure IA):
1. Construct carrying the bovine growth hormone (BGH) PolyA downstream of the
SA_GFP (SA_GFP_BGH);
2. Construct carrying the Woodchuck hepatitis virus post-transcriptional
regulatory
element (WPRE) downstream of the SA_GFP and upstream of the BGH PolyA
(SA_G F P_WP RE);
3. Construct containing a splice donor downstream of the SA_GFP cassette
(SA_GFP_SD) to obtain a fusion transcript including the corrected sequence and
endogenous RAG1 followed by the 3' UTR sequence;
4. Construct with the same endogenous RAG1 3'UTR following the SA_GFP cassette
(SA_GFP_3'UTR);
5. Construct with the SA_GFP cassette followed by the endogenous RAG1 3'UTR
and BGH PolyA (SA_GFP_3'UTR_BGH);
6. Construct with the SA_GFP cassette followed by the internal ribosome entry
site
sequence (IRES), a clinically compatible selector (COterminal truncated low0
affinity NGFR receptor, hereafter named NGFR) and the BGH PolyA sequences
(SA_GFP_IRES_NGFR_BGH) ¨ this strategy might allow the enrichment of edited
cells by the NGFR selector and the improvement of GFP expression through the
IRES and mRNA stabilization;
7. Construct with the SA_GFP cassette followed by the IRES, a peptide sequence
rich in proline (P), glutamic acid (E), serine (S), and threonine (T) (PEST)
and the
splice donor sequence (SA_GFP_IRES_PEST_SD) ¨ this construct will result in a
fusion transcript including the corrected sequence and the endogenous RAG1
followed by the 3' UTR sequence (it is expected that the endogenous RAG1
protein
will be destabilized by the PEST signal peptide via proteasome degradation;
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8. Construct with GFP expression driven by a PGK promoter as an internal
positive
control (PGK_GFP-BGH).
To screen the donors described above, NALM6 cells were transfected with guide
9 and Cas9
as an RNP (25 pmol) and donors as linearized DNA fragments (1600 ng), and then
kept in
culture with RPM! and 10% FBS. To synchronize cell cycles at GO/G1 phase when
the RAG1
gene is mainly expressed, cells were serum starved 16 days after the
transfection (Figure
11B).
We evaluated GFP expression as the percentage of GFP+ cells and GFP mean
fluorescence
intensity (MP!) by flow cytometry over time. The proportion of GFP+ cells was
low in all
conditions as expected because NALM6 are poorly permissive to the editing. We
confirmed
data described in Figure 3 showing that cells edited with SA_GFP_SD and
SA_GFP_3'UTR
constructs have a lower MFI than that obtained by SA_GFP_BGH (Figure 11C).
Moreover,
SA_GFP_IRES_NGFR_BGH and SA_GFP_IRES_PEST_SD did not ameliorate GFP
expression compared to other constructs (Figure 11C).
We analyzed the GFP expression 4, 5 and 7 days after serum starvation to
evaluate the
modulation of transgene expression when regulated by the RAG1 promoter. We
found that all
the donors carrying the 3'UTR or using the endogenous 3'UTR (by SD sequence)
resulted in
a modulation of GFP expression upon starvation (Figure 11D).
Editing enhancer effects on HDR efficiency of RAGI locus and T cell
differentiation potential
To further understand the efficacy of the gene editing approach to correct
RAG1 defects, we
exploited a novel organoid platform, referred to as artificial thymic organoid
(ATO) based on
the aggregation of DLL4 expressing stromal cell line (MS5-hDLL4) with CD34+
cells isolated
from bone marrow or mobilized peripheral blood. The ATO platform (Seet et al.
(2017) Nat
Methods) is a suitable tool to study the first steps of human T cell
differentiation. We adopted
this platform to assess the impact of the gene editing procedure on T cell
differentiation and
to evaluate the extent to which precise correction allows the overcoming of a
T cell
differentiation block.
To this end, we set up and optimized the ATO system using CD34+ cells obtained
from healthy
donor (HD) mobilized peripheral blood (MPB) or bone marrow (BM). One day after
editing,
CD34+ cells were aggregated with MS5-hDLL4 cells and kept in culture for 4 to
7 weeks to
assess the T cell differentiation potential and the editing efficiency (Figure
12). ATOs
generated with gene edited CD34+ cells showed lower cell viability as compared
to ATO
containing untreated CD34+ cells.
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To overcome the high toxicity likely caused by the exacerbated p53 response
and at the same
time to enhance HDR efficiency, we tested the effect of gene editing enhancer
compounds: to
this end we exploited the messenger RNA for the dominant negative p53 GSE56
with or
without Ad5-E4orf6/7, or Ad5-E4orf6/7 alone during the editing procedure. Ad5-
E4orf6/7 is an
adenoviral protein known as a helper in Ad-AAV co-infection, which interacts
with several
components involved in survival and cell cycle.
We electroporated CD34+ cells in the presence of gene editing enhancers: GSE56
or Ad5-
E4orf6/7 alone or the combination of GSE56 and Ad5-E4orf6/7 (COMBO). Cells
were then
transduced with AAV6 vectors: the corrective donor vector carrying the codon
optimized RAG1
downstream of the splice acceptor (SA) and followed by the BGH polyA
(SA_coRAG1_BGH
polyA) or the AAV6 vector carrying the PGK_GFP_BGHpolyA to track edited cells
in HPSC
cell subsets (Figure 12A). Seven days after gene editing, HDR efficiency was
assessed by
ddPCR for CD34+ cells edited with SA-coRAG1-BGHpolyA, while by flow cytometry
for 0D34+
cells edited with PGK_GFP_BGHpolyA. In the presence of the corrective donor,
molecular
analysis revealed a significant increase of the frequency of edited alleles in
the gene editing
condition performed in the presence of GSE56+Ad5-E4orf6/7 (COMBO) (Figure
12B).
Remarkably, CD34+ cells undergoing gene editing with AAV6 PGK_GFP_BGHpolyA
revealed
a frequency of 40% of GFP positive cells within the most primitive HSPC subset
(CD133+
CD90+) (Figure 12C).
Moreover, we performed multiparametric analysis of MPB or BM HSPC compositions
before
(day 0) and after gene editing (day 4) (Figure 12D). We confirmed previous
data (Figure 10)
showing a redistribution of HSPC subpopulations mainly due to the expansion
protocol. In
untreated and edited CD34+ cells at day 4, we observed a relative expansion of
hematopoietic
stem cells (HSC), multipotent progenitors (M PP) and multilymphoid progenitors
(MLP) at the
expense of common myeloid progenitors (CMP), indicating that gene editing
protocols using
GSE56+Ad5-E4orf6/7 (COMBO) preserve stemness in the composition (Figure 120).
After 24 hours from gene editing (at day 4), CD34+ cells were washed, counted
and seeded
in the presence of MS5-hDLL4 to form thymic organoids to follow T cell
differentiation for 4-7
weeks. Starting from the fourth week after the seeding, ATOs were dissociated,
and bulk cells
edited with the corrective donor were analyzed for HDR efficiency by molecular
analysis
(ddPCR), while cells edited with pGK_GFP_BGHpolyA AAV6 vector were analyzed by
flow
cytometry to detect the frequency of GFP+ cells in different T cell subsets.
Evaluation of AT0s,
showed an improvement of organoid morphology in the presence of the combined
action of
GSE56+E4orf6/7 (Figure 13A). This finding was confirmed by the increased
number of cells
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harvested from ATOs seeded with 0D34+ edited with Ad5-E4orf6/7 and reaching
the highest
values with the COMBO treatment (Figure 13B).
The molecular analysis of HDR frequency in T cells differentiated from CD34+
edited with
SA_coRAG1_BGHpolyA further confirmed the synergistic effect of GSE56+Ad5-
E4orf6/7
revealing the higher proportion of edited alleles in the COMBO condition as
compared to
others (Figure 13C). Flow cytonnetric analysis of double negative (DN), double
positive (DP),
single positive (SP) T cells obtained from the ATO seeded with CD34+ cells
edited and
transduced with the AAV6 PGK_GFP_BGHpolyA showed the highest frequency of GFP+
cells
in the COMBO condition (Figure 130). The synergistic effect of GSE56+Ad5-
E4orf6/7 was
more evident in TCRa/r3+ cell subset, a relevant subpopulation absent in RAG1-
deficient
patients.
Overall, these data indicate that the use of gene editing enhancers
dramatically enhance HDR
editing efficiency in CD34+ cells while preserving their ability to
differentiate towards T cell
lineage.
Material and methods
Donor constructs
The cloning of plasmids was performed using general molecular biology
techniques. Briefly,
plasmids were digested using restriction enzymes (New England BioLabs) and
correct
fragments were separated and purified by agarose gel electrophoresis.
Fragments were
inserted into a dephosphorylated linearized backbone with either Quick Ligase
or T4 Ligase
after purification with QIAquick PCR Purification Kit (QIAGEN). After
ligation, TOP10
chemically competent E. Coll bacteria were transformed and plated on plates
containing
antibiotics. Plasmid DNA was extracted and purified with Wizard Plus SV
Minipreps DNA
Purification System (Promega) and EndoFree Plasmid Maxi Kit (QIAGEN). Colonies
were
screened with control digestions and sequenced. Sequences of the further
inserts are shown
below:
AAV6 vector carrying SA_GFP_3'UTR_BGH, guide 9:
INSERT
tgagcacacagttattacttggaaattgtgtacagactaagttgaagatgttaggagggaagattgtgggccaagtaac
ggggtgt
atgtgtgtgggtatagggtgggcagctgggatggaaatggggggctgctgctgctgctgcaccctggcctcctgaacta
atgatat
cactcaccagaaactactgttcctgcactgtccaagccaccccaaactagtttgtcaaaatgaatctgtgctgtgtgga
gggaggc
acgcctgtagctctgatgtcagatggcaatgtgaattcctgacctcttctcttcctcccacaggccgccaccatggtga
gcaagggc
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gaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccg
gcga
gggcg
agggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccacc
ctcgtgaccaccctgacctacg gcgtgcagtgcttcagtcg ctaccccg accacatgaag
cagcacgacttcttcaagtccgcca
tgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagtt
cga
gggcg
acaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctgg
agtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccg
cca
caacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtg
ctgctgcc
tgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggag
ttc
gtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaactggatccgtagggcaaccacttatgagtt
ggifitt
gcaattgagtttccctctgggttgcattgagggcttctcctagcaccctttactgctgtgtatggggcttcaccatcca
agaggtggtag
gttggagtaagatgctacagatgctctcaagtcaggaatagaaactgatgagctgattgcttgaggcttttagtg
agttccgaaaa
gcaacaggaaaaatcagttatctg
aaagctcagtaactcagaacaggagtaactgcaggggaccagagatgagcaaag atc
tgtgtgtgttggggagctgtcatgtaaatcaaagccaaggttg
tcaaagaacagccagtgaggccaggaaagaaattg gtcttgt
ggttttcatttttttcccccttgattgattatattttgtattgagatatgataagtgccttctatttcatttttgaata
attcttcatttttataattttac
atatcttggcttgctatataagattcaaaagagcttlitaaattlitctaataatatcttacatttgtacagcatg
atgacctttacaaagtg
ctctcaatgcatttacccattcgttatataaatatgttacatcaggacaactligagaaaatcagtcclittttatgtt
taaattatgtatcta
ttgtaaccttcagagtttaggaggtcatctgctgtcatggatttttcaataatgaatttagaatacacctgttagctac
agttagttattaa
atcttctgataatatatgtttacttagctatcagaagccaagtatgattctttatttttactifitcatttcaagaaat
ttagagificcaaattta
gagclictgcatacagtcttaaagccacagaggcttgtaaaaatataggttagcttgatgtctaaaaatatatttcatg
tcttactg aa
acattttgccagactttctccaaatg aaacctgaatcaatttttctaaatctaggtttcatag
agtcctctcctctgca atgtgttattctttct
ataatgatcagtttactttcagtggattcagaattgtgtagcaggataaccttgtatttttccatccgctaagtttaga
tggagtccaaac
gcagtacagcagaagagttaacatttacacagtgctttttaccactgtggaatgttttcacactcatttttccttacaa
caattctgagg
agtaggtgttgttattatctccatttgatgggggtttaaatgatttgctcaaagtcatttaggggtaataaatacttgg
cttggaaatttaa
cacagtccttttgtctccaaagcccttcttctttccaccacaaattaatcactatgtttataaggtagtatcagaattt
ttttaggattcaca
actaatcactatagcacatgaccttggg
attacatttttatggggcaggggtaagcaagtlittaaatcatttgtgtgctctggctcttttg
atagaagaaagcaacacaaaagctccaaagggccccctaaccctcttgtggctccagttatttggaaactatgatctgc
atcctta
ggaatctgggatttgccagttgctggcaatgtagagcaggcatggaattttatatgctagtgagtcataatgatatgtt
agtgttaatta
gttttttcttcctttgattttattggccataattgctactcttcatacacagtatatcaaagagcttgataatttagtt
gtcaaaagtgcatcg
gcgacattatctttaattgtatgtatttggtgclicttcagggattgaactcagtatctttcattaaaaaacacag
caglittccttgcttlita
tatg ca g aatatcaaagtcatttctaatttagttgtcaaaaacatatacatattttaacattagttlltttg
aaaactcttg gttttgtttttttgg
aaatgagtgggccactaagccacactttcccttcatcctgcttaatccttccagcatgtctctgcactaataaacagct
aaattcacat
aatcatcctatttactgaagcatggtcatgctggtttatagattfittacccatttctactcffittctctattggtgg
cactgtaaatactttcc
agtattaaattatcclittctaacactgtaggaactattttgaatgcatgtgactaagagcatgatttatagcacaacc
litccaataatc
ccttaatcag atcacattttgataaaccctg gg aacatctgg ctg cag gaatttcaatatgta
gaaacgctgcctatg gttttttgccct
tactgttgagactgcaatatcctagaccctagttttatactagagttttatttttagcaatgcctattgcaagtgcaat
tatatactccagg
gaaattcaccacactgaatcg ag catttgtgtgtgtatgtgtgaagtatatactg gg
acttcagaagtgcaatgtatttttctcctgtg a
aacctgaatctacaagttttcctgccaag ccactcag gtgcattg cag gg accagtgataatg g ctg
atgaaaattgatgattggtc
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agtgaggtcaaaaggagccttgggattaataaacatgcactgagaagcaagaggaggagaaaaagatgtctlittcttc
caggt
gaactggaatttagttttgcctcagatttttttcccacaagatacagaagaagataaagatttttttggttgagagtgt
gggtcttgcatt
acatcaaacagagttcaaattccacacagataagaggcaggatatataagcgccagtggtagttgggaggaataaacca
ttatt
tggatgcaggtggffittgattgcaaatatgtgtgtgtcttcagtgattgtatgacagatgatgtattcttttgatgtt
aaaagattttaagta
agagtagatacattgtacccattttacattttcttattttaactacagtaatctacataaatatacctcagaaatcatt
tttggtgattatttttt
gttttgtagaattgcacttcagtttattttcttacaaataaccttacattttgtttaatggcttccaagagccttattt
tifttgtatttcagagaa
aattcaggtaccaggatgcaatggatttatttgattcaggggacctgtgtttccatgtcaaatgttttcaaataaaatg
aaatatgagtt
tcaatactlittatattttaatatttccattcattaatattatggttattgtcagcaattttatgtttgaatatttgaa
ataaaagtttaagatttga
aaatggtatgtattataatttctattcaaatattaataataatattgagtgcagcatttctaggatcctaaactgtgcc
ttctagttgccag
ccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgccctttcctaataaaatg
aggaaattgc
atcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagac
aatag
caggcatgctggggatgcggtgggctctatggtctagatggcagtggccggtggggacagggctgagccagcaccaacc
act
cagcctttgagatcccgaggctggtctactgctgagaccilttgttagaagagaggagatcaagcatttgcaaggtttc
tgagtgtc
aaaatatgaatccaagataactctttcacaatcctaacttcatgctgtctacaggtccatattttagcctgctttctcc
atgttcatccga
aaagaaagaaaagctaagggtggtggtcatatttgaaattagccagatcttaaglitttctgggggaaatttagaagaa
aatatgg
aaaagtgactatgagcaca
HA Left
tgagcacacagttattacttggaaattgtgtacagactaagttgaagatgttaggagggaagattgtgggccaagtaac
ggggtgt
atgtgtgtgggtatagggtgggcagctgggatggaaatggggggctgctgctgctgctgcaccctggcctcctgaacta
atgatat
cactcaccagaaactactgttcctgcactgtccaagccaccccaaactagtttgtcaaaatgaatctgtgctgtgtgga
gggaggc
acgcctgtagctctgatgtcagatggcaatgt
Splice Acceptor
ctgacctcttctcttcctcccacag
KOZAK
gccgccaccatg
GFP
atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccaca
agt
tcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagct
gcc
cgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagtcgctaccccgaccacatgaagcag
cacg
acttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagac
ccgc
gccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaaca
tc
ctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaagg
tg
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aacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcg
ac
gg ccccgtgctgctg cctg acaaccactacctgag cacccagtccg ccctgagcaaagaccccaacg ag
aagcg cgatcac
atggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa
3' U T R
gtagggcaaccacttatgagttggffittgcaattgagtttccctctgggttgcattgagggcttctcctagcaccctt
tactgctgtgtat
ggggcttcaccatccaagaggtggtaggttggagtaagatgctacagatgctctcaagtcaggaatagaaactgatgag
ctgatt
gcttgaggcttttagtgagttccgaaaagcaacaggaaaaatcagttatctgaaagctcagtaactcagaacaggagta
actgc
aggggaccagagatgagcaaagatctgtgtgtgttggggagctgtcatgtaaatcaaagccaaggttgtcaaagaacag
ccag
tgaggccaggaaagaaattggtcttgtggttttcattlitttcccccttgattgattatattttgtattgagatatgat
aagtgccttctatttca
tlittgaataattcttcatttttataattttacatatcttg gcttgctatataag attcaaaa gag
cttlitaaattffictaataatatcttacattt
gtacagcatgatgacctttacaaagtgctctcaatgcatttacccattcgttatataaatatgttacatcaggacaact
ttgagaaaat
cagtcctffittatgtttaaattatgtatctattgtaaccttcagagtttaggaggtcatctgctgtcatggatttttc
aataatgaatttagaa
tacacctgttagctacagttagttattaaatcttctgataatatatgtttacttagctatcagaagccaagtatgattc
tttatttttactttttc
atttcaagaaatttagagtttccaaatttagagcttctgcatacagtcttaaagccacagaggcttgtaaaaatatagg
ttagcttgat
gtctaa aaatatatttcatgtcttactgaaacattttgccagactttctccaaatg
aaacctgaatcaatttttctaaatctag gtttcatag
agtcctctcctctgcaatgtgttattctttctataatgatcagtttactttcagtggattcagaattgtgtag
caggataaccttgtattlitcc
atccgctaagtttagatggagtccaaacgcagtacagcagaagagttaacatttacacagtgctttttaccactgtgga
atgttttca
cactcatttttccttacaacaattctgaggagtaggtgttgttattatctccatttgatgggggtttaaatgatttgct
caaagtcatttagg
ggtaataaatacttggcttggaaatttaacacagtcclittgtctccaaagcccttcttctttccaccacaaattaatc
actatgtttataa
ggtagtatcagaattffittaggattcacaactaatcactatagcacatgaccttgggattacatttttatggggcagg
ggtaagcaag
tttttaaatcatttgtgtgctctggctcttttgatagaagaaagcaacacaaaagctccaaagggccccctaaccctct
tgtggctcca
gttatttggaaactatgatctgcatccttaggaatctgggatttgccagttgctggcaatgtagagcaggcatggaatt
ttatatgctag
tgagtcataatgatatgttagtgttaattagtffittcttcctttgattttattggccataattgctactcttcataca
cagtatatcaaagagct
tgataatttagttgtcaaaagtg
catcggcgacattatctttaattgtatgtatttggtgcttcttcagggattgaactcagtatctttcatta
aaaaacacagcagttttccttgctttttatatgcagaatatcaaagtcatttctaatttagttgtcaaaaacatataca
tattttaacatta
gffittttgaaaactcttggttttgtttttttggaaatgagtgggccactaagccacactttcccttcatcctgcttaa
tccttccagcatgtct
ctgcactaataaacagctaaattcacataatcatcctatttactgaagcatggtcatgctggtttatagattttttacc
catttctactctlit
tctctattggtgg cactgtaaatactttccagtattaaattatccttttctaacactgtagg aactattttgaatg
catgtg actaagagca
tgatttatagcacaacctttccaataatcccttaatcagatcacattttgataaaccctgg
gaacatctggctgcaggaatttcaatat
gtagaaacgctgcctatggtiltttgcccttactgttgagactgcaatatcctagaccctagttttatactagagtttt
attlltagcaatgc
ctattgcaagtgcaattatatactccagggaaattcaccacactgaatcgagcatttgtgtgtgtatgtgtgaagtata
tactggg act
tcagaagtgcaatgtatttttctcctgtg aaacctgaatctacaagttttcctgccaag
ccactcaggtgcattgcagggaccagtg a
taatggctgatgaaaattgatgattggtcagtgaggtcaaaaggagccttgggattaataaacatgcactgagaag
caagagga
ggagaaaaagatgtctllttcttccaggtgaactggaatttagttttgcctcagatttttttcccacaagatacagaag
aagataaaga
tffitttggttgagagtgtgggtcttgcattacatcaaacagagttcaaattccacacagataagaggcag
gatatataagcgccagt
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ggtagttgggaggaataaaccattatttggatgcaggtggthttgattgcaaatatgtgtgtgtcttcagtgattgtat
gacagatgat
gtattcttttgatgttaaaagattttaagtaagagtagatacattgtacccattttacattttcttattttaactacag
taatctacataaatat
acctcagaaatcatttttggtgattattttttgttttgtagaattgcacttcagtttattttcttacaaataaccttac
attttgtttaatggcttcc
aagagccttthtttttttgtatttcagagaaaattcaggtaccaggatgcaatggatttatttgattcaggggacctgt
gtttccatgtcaa
atgttttcaaataaaatgaaatatgagtttcaatactttttatattttaatatttccattcattaatattatggttatt
gtcagcaattttatgtttg
aatatttgaaataaaagtttaagatttgaaaatggtatgtattataatttctattcaaatattaataataatattgagt
gcagcatt
BGH
actgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactccca
ctgccctttcct
aataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaa
ggggg
aggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgg
HA Right
atggcagtggccggtggggacagggctgagccagcaccaaccactcagcctttgagatcccgaggctggtctactgctg
agac
cttttgttagaagagaggagatcaagcatttgcaaggtttctgagtgtcaaaatatgaatccaagataactctttcaca
atcctaactt
catgctgtctacaggtccatattttagcctgctttctccatglicatccgaaaagaaagaaaagctaagggtggtggtc
atatttgaa
attagccagatcttaagtttttctgggggaaatttagaagaaaatatggaaaagtgactatgagcaca
AAV6 vector carrying SA_GFP_IRES_NGFR_BGH-RAG1, guide 9:
INSERT
tgagcacacagttattacttggaaattgtgtacagactaagttgaagatgttaggagggaagattgtgggccaagtaac
ggggtgt
atgtgtgtgggtatagggtgggcagctgggatggaaatggggggctgctgctgctgctgcaccctggcctcctgaacta
atgatat
cactcaccagaaactactgttcctgcactgtccaagccaccccaaactagtttgtcaaaatgaatctgtgctgtgtgga
gggaggc
acgcctgtagctctgatgtcagatggcaatgtgaattcctgacctcttctcttcctcccacaggccgccaccatggtga
gcaagggc
gaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccg
gcga
gggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggccc
acc
ctcgtgaccaccctgacctacggcgtgcagtgcttcagtcgctaccccgaccacatgaagcagcacgacttcttcaagt
ccgcca
tgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagtt
cga
gggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctg
g
agtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccg
cca
caacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctg
cc
tgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggag
ttc
gtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaactggatccgaattaactcgaggaattccgc
ccctc
tccctcccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttcc
accatattgcc
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gtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattctaggggtclitcccctctcgcca
aaggaatgc
aaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttg
caggcag
cggaaccccccacctggcgacaggtgcctctgcggccaaaagccaacgtgtataagatacacctgcaaaggcggcacaa
cc
ccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaag
gatgcc
cagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaa
acgtctag
gccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataatatggccacaaccatgggagctggtgcta
ccggc
agagctatggatggacctagactgctgctcctgctgctgctcggagtttctcttggcggagccaaagaggcctgtccta
ccggcct
gtatacacactctggcgagtgctgcaaggcctgcaatcliggagaaggcgtggcacagccttgcggcgctaatcagaca
gtgtg
cgagccttgcctggacagcgtgacctttagcgacgtggtgtctgccaccgagccatgcaagccttgtaccgagtgtgtg
ggcctg
cagagcatgtctgccccttgtgtggaagccgacgatgccgtgtgtagatgcgcctacggctactaccaggacgagacaa
cagg
cagatgcgaggcctgtagagtgtgtgaagccggctctggactggtgttcagctgccaagacaagcagaacaccgtgtgc
gagg
aatgccccgatggcacctatagcgacgaggccaaccatgtagatccctgcctgccttgtactgtgtgcgaagataccga
gcggc
agctgcgcgagtgtacaagatgggctgatgccgagtgcgaagagatccccggcagatggatcaccagaagcacacctcc
ag
agggcagcgatagcacagccccttctacacaagagcccgaggctectcctgagcaggatctgattgcctctacagtggc
cggc
gtggtcacaacagtgatgggatcttctcagcccgtggtcaccagaggcaccaccgacaatctgatccccgtgtactgta
gcatcct
ggccgccgtggttgtgggactcgtggcctatatcgccttcaagcggtggaaccggggcatcctgtaatgatctagcaac
ccgctg
atcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctccmcgtgccttccttgaccctggaaggtg
ccactccca
ctgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggg
gcaggacag
caagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggtctagaatggcagtggccgg
tgg
ggacagggctgagccagcaccaaccactcagcctttgagatcccgaggctggtctactgctgagaccttttgttagaag
agagg
agatcaagcatttgcaaggtttctgagtgtcaaaatatgaatccaagataactctttcacaatcctaacttcatgctgt
ctacaggtcc
atattttagcctgctttctccatgttcatccgaaaagaaagaaaagctaagggtggtggtcatatttgaaattagccag
atcttaagttt
ttctgggggaaatttagaagaaaatatggaaaagtgactatgagcaca
HA Left
tgagcacacagttattacttggaaattgtgtacagactaagttgaagatgttaggagggaagattgtgggccaagtaac
ggggtgt
atgtgtgtgggtatagggtgggcagctgggatggaaatggggggctgctgctgctgctgcaccctggcctcctgaacta
atgatat
cactcaccagaaactactgttcctgcactgtccaagccaccccaaactagffigtcaaaatgaatctgtgctgtgtgga
gggaggc
acgcctgtagctctgatgtcagatggcaatgt
Splice Acceptor
ctgacctcttctcttcctcccacag
KOZAK
gccgccaccatg
GFP
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atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccaca
agt
tcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagct
gcc
cgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgettcagtcgctaccccgaccacatgaagcag
cacg
acttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttettcaaggacgacggcaactacaagac
ccgc
gccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaaca
tc
ctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaagg
tg
aacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcg
ac
ggccccgtgctgctgcctgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatc
ac
atggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa
IRES
gaattaactcgaggaattccgCcectctccctcccccccccctaacgttactggccgaagccgcttggaataaggccgg
tgtgcg
tttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttga
cgagcattctagg
ggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaa
gacaaac
aacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccaacgtgt
ataa
gatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcct
caagc
gtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgct
ttacatg
tgtttagtcgaggttaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataat
atggcca
caacc
NGFR
atgggagctggtgctaccggcagagctatggatggacctagactgctgctcctgctgctgctcggagtttctcttggcg
gagccaa
agaggcctgtcctaccggcctgtatacacactctggcgagtgctgcaaggcctgcaatcttggagaaggcgtggcacag
ccttg
cggcgctaatcagacagtgtgcgagccttgcctggacagcgtgacctttagcgacgtggtgtctgccaccgagccatgc
aagcc
ttgtaccgagtgtgtgggcctgcagagcatgtctgccccttgtgtggaagccgacgatgccgtgtgtagatgcgcctac
ggctacta
ccaggacgagacaacaggcagatgcgaggcctgtagagtgtgtgaagccggctctggactggtgttcagctgccaagac
aag
cagaacaccgtgtgcgaggaatgccccgatggcacctatagcgacgaggccaaccatgtagatccctgcctgccttgta
ctgtg
tgcgaagataccgagcggcagctgcgcgagtgtacaagatgggctgatgccgagtgcgaagagatccccggcagatgga
tc
accagaagcacacctccagagggcagcgatagcacagccccttctacacaagagcccgaggctcctcctgagcaggatc
tg
attgcctctacagtggccggcgtggtcacaacagtgatgggatcttctcagcccgtggtcaccagaggcaccaccgaca
atctg
atccccgtgtactgtagcatcctggccgccgtggttgtgggactcgtggcctatatcgccttcaagcggtggaaccggg
gcatcct
gtaa
BGH
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ctgtgccttctagttgccagccatctgttglitgcccctcccccgtgccttccttgaccctggaaggtgccactcccac
tgtcctttccta
ataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaag
gggga
ggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgg
HA Right
atggcagtggccggtggggacagggctgagccagcaccaaccactcagcctttgagatcccgaggctggtctactgctg
agac
cttttgttagaagagaggagatcaagcatttgcaaggtttctgagtgtcaaaatatgaatccaagataactctttcaca
atcctaactt
catgctgtctacaggtccatattttagcctgctttctccatgttcatccgaaaagaaagaaaagctaagggtggtggtc
atatttgaa
attagccagatcttaagthttctgggggaaatttagaagaaaatatggaaaagtgactatgagcaca
AAV6 vector carrying SA_GFP_IRES_PEST_SD-RAG1, guide 9:
INSERT
tgagcacacagttattacttggaaattgtgtacagactaagttgaagatgttaggagggaagattgtgggccaagtaac
ggggtgt
atgtgtgtgggtatagggtgggcagctgggatggaaatggggggctgctgctgctgctgcaccctggcctcctgaacta
atgatat
cactcaccagaaactactgttcctgcactgtccaagccaccccaaactagtttgtcaaaatgaatctgtgctgtgtgga
gggaggc
acgcctgtagctctgatgtcagatggcaatgtgaattcctgacctcttctcttcctcccacaggccgccaccatggtga
gcaagggc
gaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccg
gcga
gggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggccc
acc
ctcgtgaccaccctgacctacggcgtgcagtgcttcagtcgctaccccgaccacatgaagcagcacgacttcttcaagt
ccgcca
tgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagtt
cga
gggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctg
g
agtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccg
cca
caacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctg
cc
tgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggag
ttc
gtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaactggatccgaattaactcgaggaattccgc
ccctc
tccctcccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttcc
accatattgcc
gtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattctaggggtctttcccctctcgcca
aaggaatgc
aaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttg
caggcag
cggaaccccccacctggcgacaggtgcctctgcggccaaaagccaacgtgtataagatacacctgcaaaggcggcacaa
cc
ccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaag
gatgcc
cagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtglitagtcgaggttaaaaa
acgtctag
gccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataatatggccacaaccatgaggaccgaggccc
ccg
agggcaccgagagcgagatggagacccccagcgccatcaacggcaaccccagctggcacccggatccaggtaagttcta
g
aatggcagtggccggtggggacagggctgagccagcaccaaccactcagcctttgagatcccgaggctggtctactgct
gaga
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ccttttgttagaagagaggagatcaagcatttgcaagglitctgagtgtcaaaatatgaatccaagataactctttcac
aatcctaac
ttcatgctgtctacaggtccatattttagcctgctlictccatgttcatccgaaaagaaagaaaagctaagggtggtgg
tcatatttga
aattagccagatcttaagtttttctgggggaaatttagaagaaaatatggaaaagtgactatgagcaca
HA Left
tgagcacacagttattacttggaaattgtgtacagactaagttgaagatgttaggagggaagattgtgggccaagtaac
ggggtgt
atgtgtgtgggtatagggtgggcagctgggatggaaatggggggctgctgctgctgctgcaccctggcctcctgaacta
atgatat
cactcaccagaaactactgttcctgcactgtccaagccaccccaaactagtttgtcaaaatgaatctgtgctgtgtgga
gggaggc
acgcctgtagctctgatgtcagatggcaatgt
Splice Acceptor
ctgacctcttctcttcctcccacag
KOZAK
gccgccaccatg
GFP
atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccaca
agt
tcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagct
gcc
cgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagtcgctaccccgaccacatgaagcag
cacg
acttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagac
ccgc
gccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaaca
tc
ctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaagg
tg
aacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcg
ac
ggccccgtgctgctgcctgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatc
ac
atggtcctgctggagttcgtg accg ccg ccgg gatcactctcg gcatg gacgagctgtacaagtaa
!RES
gaattaactcgaggaattccgCcectctccctcccccccccctaacgttactggccgaagccgcttggaataaggccgg
tgtgcg
tttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttga
cgagcattctagg
ggtclitcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaa
gacaaac
aacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccaacgtgt
ataa
gatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcct
caagc
gtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgct
ttacatg
tgtttagtcgaggttaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataat
atggcca
caacc
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PEST
atgaggaccgaggcccccgagggcaccgagagcgagatggagacccccagcgccatcaacggcaaccccagctggcac
Splice Donor
aggtaagt
HA Right
atggcagtggccggtggggacagggctgagccagcaccaaccactcagcctttgagatcccgaggctggtctactgctg
agac
ctfttgttagaagagaggagatcaagcatttgcaaggtttctgagtgtcaaaatatgaatccaagataactctttcaca
atcctaactt
catgctgtctacaggtccatattttagcctgctttctccatgttcatccgaaaagaaagaaaagctaagggtggtggtc
atatttgaa
attagccagatcttaagtttttctgggggaaatttagaagaaaatatggaaaagtgactatgagcaca
AA V6 gene-editing protocol in cell lines
5x105 cells per well were electroporated (Lonza, SF Cell line 4D Nucleofector
X Kit, program
FF120 for K562 or program DS100 for NALM6) with either plasmids or RNPs. Donor
DNA was
delivered by electroporation as fragment plasmid spanning the region between
the left and
right homology arms at a dose of 1600 ng.
CD34+ cells
Human MPB or BM CD34+ cells were obtained from Lonza and stimulated in
StemSpan
medium supplemented with penicillin/streptomycin antibiotics and early-acting
cytokines:
Stem cell factor (SCF) 300 ng/ml, Flt3 ligand (Flt3-L) 300 ng/ml,
Thrombopoietin (TPO) 100
ng/ml, StemRegenin1 (SRI) (1 pM) and 16,16-dimethyl prostaglandin E2 (dmPGE2)
(10pM),
UM171 35nM.
AA V6 gene-editing protocol in CD34+ cells
After 3 days of expansion, 2-5x105 CD34+ cells per condition were
electroporated (Lonza, P3
Primary Cell 4DNucleofector X Kit, CD34+ program) with RNPs, GSE56 mRNA (3
ug/test),
Ad5-E4orf6/7 (1.5ug/test) or GSE56+Ad5-E4orf6/7 as fusion protein with P2A
self cleaving
peptide (5ug/test). 15 minutes after electroporation, CD34+ cells were
infected with AAV6 at
104 Vg/cell and kept in culture with StemSpan medium supplemented with
penicillin/streptomycin antibiotics and early-acting cytokines: Stem cell
factor (SCF) 300 ng/ml,
Flt3 ligand (Flt3-L) 300 ng/ml, Thronnbopoietin (TPO) 100 ng/ml ,
StennRegenin1 (SR1) (1 pM)
and UM171 35 nM.
Flow cytometry analysis (FACS) and sorting
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For the analysis of GFP expression, unstained and single-stained cells or
compensation beads
were used as negative and positive controls. For apoptosis/necrosis detection,
cells were
stained with 7-Aminoactinomycin D (7-AAD, BD Pharming). CD34+ cells were
stained with
phycoerythrin cyanine 7 (PECy7) CD34 (Clone: AC136, Miltenyi Biotec),
phycoerythrin (PE)
CD133 (Miltenyi Biotec) allophycocyanin (APC) CD90 (BD Biosciences). Cell
sorting on
CD133/CD90 edited cells was performed using MoFlo XDP Cell Sorter (Beckman
Coulter).
Analysis of HSPC composition of MPB/BM-CD34+ cells was performed according to
the
protocol in (Basso-Ricci et al. (2017) Cytom Part A. 91: 952-65). Briefly,
1.5x105 cells were
labeled with fluorescent antibodies against CD3, CD56, CD14, CD61/41, CD135,
CD34,
CD45RA (Biolegend) and CD33, CD66b, CD38, CD45, CD90, CD10, CD11c, CD19, CD7,
and CD71 (BD Biosciences). All samples were acquired through BD LSR-Fortessa
(BD
Bioscience) cytofluorimeter after Rainbow bead (Spherotech) calibration and
raw data were
collected through DIVA software (BD Biosciences).
T cell differentiation was analyzed after cell harvesting from ATOs by flow
cytometry using the
following mAb: TCRab APC (Cl. IP26, eBioscience), CD4 Alexa Fluor 700 (Cl.
OKT4,
eBioscience), CD19 PerCP-Cy5.5 (cl. HIB19, Biolegend), CD56 FITC (cl. MEM-188,
Biolegend), CD8a PE/Dazzle (Cl. RPA-T8, Biolegend), CD45 V500 (Cl. HI30, BD
Biosciences),
CD3 BV421 (cl. UCHT1, BD Biosciences), CD8b PE (cl. 2ST8.5H7, BD Biosciences)
LIVE/DEADTM Fixable Yellow Dead Cell Stain Kit (Invitrogen). All samples were
acquired
through BD Canto!! (BD Bioscience) cytofluorimeter after Rainbow bead
(Spherotech)
calibration and raw data were collected through DIVA software (BD
Biosciences).
The data were subsequently analyzed with FlowJo software Version 9.3.2
(TreeStar) and the
graphical output was automatically generated through Prism 6.0c (GraphPad
software).
Clonogenic assay
CFU-C assay was performed 24 h after editing procedure by plating 600 cells in
methylcellulose-based medium (MethoCult H4434, StemCell Technologies)
supplemented
with 100 Umi penicillin and 100 pg/ml streptomycin. Three technical replicates
were
performed for each condition. Two weeks after plating, colonies were counted
and identified
according to morphological criteria.
ATO culture system
ATOs were generated as described in Seet et al (Seet et al. (2017) Nat
Methods). Briefly, one
day after the editing procedure 5000-10000 CD34 + from BM or MPB samples
(commercially
available, Lonza) were combined with 150000 MSS-hDLL4 cells per ATO. We
normalized the
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number of "true" live 0D34+ cells according to the flow cytometry analysis
excluding dead and
CD34- cells. Each ATO (5 pl) was then plated in a 0.4 pM Millicell Transwell
insert, placed on
a well of a 6-well plate containing 1 ml complete RB27 medium supplemented
with rhIL-7 (5
ng/ml), rhFlt3-L (5 ng/ml) and 30 pM 1-ascorbic acid 2-phosphate
sesquimagnesium salt
hydrate. Each insert contained a maximum of two ATOs. Medium was changed every
3-4
days. From weeks 4 to 9, ATOs were collected by adding MACS buffer (PBS with
7.5% BSA
and 0.5 M EDTA) to each well and pipetting to dissociate the ATOs. Cells were
then
resuspended in FACS Buffer (PBS 2% FBS), counted and stained with the
following
antibodies: CD14 PE, CD45 PerCP-Cy5.5, CD1a APC, CD7 Alexa Fluor 700, CD5 PE-
Cy7,
CD34 VioBlue, CD56 FITC, CD8a APC, TCRab PerCP-Cy5.5, CD3 APC, CD4 PeVio770,
CD8b PE. Yellow live dead was used to exclude dead cells. Samples were
analyzed using
FlowJo software version 10.5.2 (FlowJo, LLC, Ashland, OR).
Digital PCR
Digital PCR (ddPCR) was performed to assess targeted integration. Briefly,
gDNA was
quantified using Nanodrop, and diluted in H20 to reach 5-10 ng per reaction (1-
2 ng/ul). It is
possible to increase the gDNA quantity per reaction but it is important to
remain below the
saturation limit of the system. ddPCR master mix was prepared by adding 11 ul
ddPCR
Supermix for Probes (no dUTP; BioRad), 1.1 ul primer mix Primer forward +
Primer reverse
(final concentration 0.9 uM) + Probe (final concentration 0.25 uM), 1.1 ul
normalizer primer
mix, 4.9 ul H20 per reaction. Finally, 17 ul of ddPCR master mix and 5 ul of
diluted gDNA were
added to each well (we included UT and H20 as negative controls, and mono- or
bi allelic
clone as positive control to validate the system). Droplets were prepared on
the BioRad
AutoDG Automated Droplet Generator and the droplet plate was sealed with foil
using BioRad
PX1 PCR Plate Sealer. The sealed plate was placed into BioRad T100 Thermal
Cycler and
we ran the appropriate PCR program. The run was read in BioRad QX200 Droplet
Reader.
Calculation copies per genome: concentration (copies/pi) gene of interest /
concentration
(copies/p1) nornnalizer gene x 2 Calculation percentage of H DR: copies per
genome x 100.
Optimized PCR program (40 cycles):
95 C x 10 min
40 x 94 x 30 sec
55 x 1 min
72 x 2 min
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98 x 10 min
4 hold
Primers and Probes used for the ddPCR assay are the following:
PGK_GFP cassette FW CAAGAGGTTGTCTGAAGGAAG
PGK_GFP cassette RV GACGTGAAGAATGTGCGAG
PGK_GFP cassette PROBE FAM CTGCTGCACCCTGGCCTCCTGAACTAA
Corrective CDS FW GTGGAACAGGTGTGATAATGAG
Corrective CDS RV GGAGGACAATCCAAGGGTAG
Corrective CDS PROBE FAM TGCTGCTGCACCCTGGCCTCCTGAA
RT-qPCR
For gene expression analyses, total RNA was extracted using RNeasy Plus Micro
Kit
(QIAGEN), according to the manufacturer's instructions and DNase treatment was
performed
using RNase-free DNase Set (QIAGEN). cDNA was synthetized with the High
Capacity cDNA
Reverse Transcription kit (Applied Biosystem). cDNA was then used for qPCR in
a Viia7 Real-
time PCR thermal cycler using Power Syber Green PCR Master Mix (Applied
Biosystems).
Data were analyzed with Viia7 Real-Time PCR software (Applied Biosystem).
Relative expression of each target gene was represented as fold changes (2-
Act) relative to
the beta-actin normalizer.
EXAMPLE 3
Results and discussion
Two further donor constructs were designed and generated:
i) a SA_coRAG1 CDS_BGHpA donor carrying the bovine growth hormone (BGH)
PolyA downstream the SA_ coRAG1CDS allowing the transcription termination of
the corrective RAG 1 CDS (Figure 14A);
ii) a SA_coRAG1 CDS_SD containing a splice donor (SD) sequence to obtain a
fusion transcript including the corrected codon optimized sequence and
endogenous RAG 1 followed by the 3' UTR sequence (Figure 14B).
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To test the two corrective donors, NALM6.Rag1K0 cells were transfected with
guide 9 and
Cas9 as RNP (50pm01) and transduced with SA_coRAG1 CDS_BGHpA or SA_coRAG1
CDS_SD AAV6 donor at two doses (104 and 5x104) (Figure 15A). As expected, we
obtained
low proportion of edited alleles in bulk edited NALM6.Rag1K0 cells due to the
low
permissiveness of NALM6 cells to HDR-mediated editing. To evaluate gene
editing efficiency
in terms of RAG1 expression and recombination activity, edited bulk
NALM6.Rag1K0 cells
were subcloned to isolate various single colonies carrying mono- or bi-allelic
editing (Figure
15A). We screened 429 clones by ddPCR and we identified 5 mono-allelic clones
edited by
SA_coRAG1 CDS_BGHpA and 11 mono-allelic clones edited by SA_coRAG1 CDS_SD.
To compare the correction efficiency of the two donors into the selected
edited clones, we
analyzed the RAG1 CDS expression by RT-qPCR and the recombination activity
assessed by
the transduction of cells with a LV carrying an inverted GFP cassette which is
recombined in
presence of a functional RAG1 protein (Liang HE, et al. Immunity. 2002;17:639-
651;
Bredemeyer AL, et al. Nature. 2006;442(7101):466-470; De Ravin SS, et al.
Blood.
2010;116:1263-1271; Lee YN, et al., J Allergy Clin lmmunol. 2014;133(4):1099-
1O).
We observed the increase of RAG1 CDS expression (Figure 15B) and recombination
activity
(Figure 15C) in the majority of clones edited by SA_coRAG1 CDS_BGHpA or
SA_coRAG1
CDS_SD AAV6 donor.
To compare the two donors in terms of impact on hematopoietic stem and
progenitor cells
(HSPC), we edited HSPC derived from the mobilized peripheral blood of HD with
guide 9 and
Cas9 as RNP (50pm01) in presence of the combination of editing enhancers
(GSE56 and Ad5-
E4orf6/7) followed by the transduction with SA_coRAG1 CDS_BGHpA or SA_coRAG1
CDS_SD AAV6 donor at three different doses.
We observed comparable editing efficiencies between HSPC edited by SA_coRAG1
CDS_BGHpA or SA_coRAG1 CDS_SD AAV6 donor, increasing according to the dose
(Figure 16A) as also confirmed by the analysis of editing efficiency in sorted
HSPC (Figure
16B). Beside the known impact of gene editing on cell growth (Figure 16C) and
clonogenic
potential (Figure 160) as compared to untreated cells, HSPC edited by
SA_coRAG1
CDS_BGHpA or SA_coRAG1 CDS_SD AAV6 donor showed similar i) kinetics of growing
(Figure 16C), ii) generation of erythroid and myeloid colonies (Figure 160),
and iii) cell subset
composition with preservation of the most primitive 0D34+ CD133+ CD90+ cells
(Figure 16E).
To further compare the two AAV6 donor constructs, we exploited the artificial
thymic organoid
(ATO) platform to differentiate edited HSPC towards the T cell lineage by
applying the protocol
previously described (Figure 12). Hematopoietic stem and progenitor cells
edited by the two
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donors similarly differentiated in early and late T cell subsets (Figure 16F)
with comparable
levels of editing efficiency in sorted double negative CD4- CD8- cells and
double positive
CD4+ CD8+ cells (Figure 16G).
Overall, these data indicate that both corrective donors are able to obtain
efficient targeting
while preserving the most primitive CD34+ CD133+ CD90+ cells subpopulation.
All publications mentioned in the above specification are herein incorporated
by reference.
Various modifications and variations of the disclosed polynucleotides,
vectors, RNAs,
methods, cells, kits, compositions, systems and uses of the invention will be
apparent to the
skilled person without departing from the scope and spirit of the invention.
Although the
invention has been disclosed in connection with specific preferred
embodiments, it should be
understood that the invention as claimed should not be unduly limited to such
specific
embodiments. Indeed, various modifications of the disclosed modes for carrying
out the
invention, which are obvious to the skilled person are intended to be within
the scope of the
following claims.
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