US Pat. No. 9,315,788

METHOD FOR THE GENERATION OF COMPACT TALE-NUCLEASES AND USES THEREOF

CELLECTIS, S.A., Paris (...

1. A compact Transcription Activator-Like Effector Nuclease (TALEN) monomer comprising:
(i) one core TALE scaffold comprising Repeat Variable Dipeptide regions (RVDs) having DNA binding specificity onto a specific
double-stranded DNA target sequence of interest; and

(ii) at least one I-TevI nuclease domain, wherein said I-TevI nuclease domain consists essentially of:
(a) amino acids 1-196 of SEQ ID NO: 20; or an amino acid sequence having at least 80%, at least 90%, or at least 95% amino
acid sequence identity with amino acids 1-196 of SEQ ID NO: 20;

(b) amino acids 1-183 of SEQ ID NO: 20; or an amino acid sequence having at least 80%, at least 90%, or at least 95% amino
acid sequence identity with amino acids 1-183 of SEQ ID NO: 20;

(c) amino acids 1-169 of SEQ ID NO: 20; or an amino acid sequence having at least 80%, at least 90%, or at least 95% amino
acid sequence identity with amino acids 1-169 of SEQ ID NO: 20;

(d) amino acids 1-148 of SEQ ID NO: 20; or an amino acid sequence having at least 80%, at least 90%, or at least 95% amino
acid sequence identity with amino acids 1-148 of SEQ ID NO: 20; or

(e) amino acids 1-123 of SEQ ID NO: 20; or an amino acid sequence having at least 80%, at least 90%, or at least 95% amino
acid sequence identity with amino acids 1-123 of SEQ ID NO: 20;

wherein said I-TevI nuclease domain is capable of cleaving DNA a few base pairs away from said double-stranded DNA target
sequence of interest when fused to the C-terminus or N-terminus of said core TALE scaffold from (i);
wherein said compact TALEN monomer is assembled to bind said target DNA sequence and cleave double-stranded DNA without requiring
dimerization.

US Pat. No. 9,044,492

METHOD FOR MODULATING THE EFFICIENCY OF DOUBLE-STRAND BREAK-INDUCED MUTAGENESIS

CELLECTIS SA, Romainvill...

1. A method for increasing double-strand break-induced mutagenesis in a cell comprising introducing into said cell:
i. at least one interfering agent, wherein said at least one interfering agent targets the sequence of SEQ ID NO: 106;
ii. at least one delivery vector comprising at least one double-strand break creating agent;
thereby obtaining a eukaryotic cell in which double-strand break-induced mutagenesis is increased.
US Pat. No. 9,982,251

LARGE VOLUME EX VIVO ELECTROPORATION METHOD

CELLECTIS S.A., (FR)

1. A method of treating vesicles with exogenous material for insertion of the exogenous material into the vesicles, consisting essentially of the steps of:a. statically retaining the vesicles and the exogenous material in a low conductivity medium, having a conductivity between 50 microSiemens/cm and 500 microSiemens/cm, in a suspension in a treatment volume in a chamber which includes parallel plate electrodes, wherein the chamber has a geometric factor (cm?1) defined by the quotient of the electrode gap squared (cm2) divided by the chamber volume (cm3) wherein said geometric factor is less than 0.1 cm?1 and greater than 0.000001 cm?1,
b. adjusting the suspension of the vesicles, the exogenous material, and the medium such that the suspension in the treatment volume in the chamber has conductivity in a range between 50 microSiemens/cm and 500 microSiemens/cm,
c. providing that the resistance of the suspension in the chamber is greater than one ohm,
d. enclosing the suspension in the chamber during treatment, wherein the treatment volume of the chamber is scalable in a range spanning 2 to 10 milliliters capacity, and
e. treating the static suspension enclosed in the chamber with one or more pulsed electric fields wherein said one or more pulsed electric fields include a rectangular voltage pulse waveform to produce a uniform pulse electric field between said parallel plate electrodes greater than 100 volts/cm and less than 5,000 volts/cm, substantially uniform throughout the treatment volume.

US Pat. No. 9,273,296

MEGANUCLEASE VARIANTS CLEAVING A DNA TARGET SEQUENCE FROM A GLUTAMINE SYNTHETASE GENE AND USES THEREOF

CELLECTIS, Romainville (...

1. A method of cleaving a DNA target sequence from a glutamine synthetase gene comprising contacting said DNA target sequence
with an I-CreI variant to thereby cleave said DNA target sequence
wherein said I-CreI variant comprises a first monomer and a second monomer which are associated to form an active form,
wherein said I-CreI variant comprises at least two substitutions in at least one of the monomers,
said first monomer has a sequence that is at least 97% identical to SEQ ID NO: 271 and said second monomer has a sequence
that is at least 97% identical to SEQ ID NO: 246 and

wherein said DNA target sequence is SEQ ID NO: 30.
US Pat. No. 9,944,702

CD33 SPECIFIC CHIMERIC ANTIGEN RECEPTORS FOR CANCER IMMUNOTHERAPY

CELLECTIS, Paris (FR)

1. A method of treating a pre-malignant condition or cancer expressing CD33, comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an engineered immune cell expressing at the cell surface membrane a CD33 specific Chimeric Antigen Receptor (CAR) comprising: (a) an extracellular ligand binding-domain comprising VH and VL from a monoclonal anti-CD33 antibody, (b) a hinge selected from Fc?RIIIa hinge, CD8? hinge, or IgG1 hinge, (c) a CD8? transmembrane domain and (d) a cytoplasmic domain comprising a CD3 ? signaling domain and a co-stimulatory domain from 4-1BB.
US Pat. No. 9,855,297

METHODS FOR ENGINEERING T CELLS FOR IMMUNOTHERAPY BY USING RNA-GUIDED CAS NUCLEASE SYSTEM

CELLECTIS, Paris (FR)

1. A method of preparing genetically modified primary T-cells for immunotherapy comprising the steps of:
(a) transfecting mRNA encoding an RNA-guided endonuclease into the primary T-cells, wherein the RNA-guided endonuclease is
expressed from the transfected m RNA;

(b) introducing a DNA vector that encodes a specific guide RNA, wherein the specific guide RNA directs the RNA-guided endonuclease
to at least one targeted locus in the T-cell genome into the primary T-cells;

(c) cleaving the at least one targeted locus in the T-cell genome with the RNA-guided endonuclease;
(d) generating a genetic modification at the site of the cleavage; and
(e) expanding the resulting genetically modified T-cells.
US Pat. No. 10,006,052

LAGLIDADG HOMING ENDONUCLEASE CLEAVING THE C-C CHEMOKINE RECEPTOR TYPE-5 (CCR5) GENE AND USES THEREOF

CELLECTIS, Paris (FR) PR...

1. A chimeric endonuclease comprising:an endonuclease domain having at least 90% identity with the amino acid sequence of SEQ ID NO:19 that cleaves a target nucleic acid sequence within the C-C chemokine receptor type 5 gene (CCR5), wherein the target nucleic acid sequence is SEQ ID NO:5; and
at least an additional protein domain selected from the group consisting of: nucleic acid binding domain, catalytic domain, terminal epitope tags and fluorescent proteins.
US Pat. No. 9,540,623

METHOD FOR INCREASING THE EFFICIENCY OF DOUBLE-STRAND-BREAK INDUCED MUTAGENESIS

CELLECTIS, Paris (FR)

1. A method for increasing double-strand-break induced mutagenesis at a genomic locus of interest in a cell comprising the
steps of:
(i) identifying at said genomic locus of interest at least one DNA target sequence cleavable by one natural or engineered
rare-cutting endonuclease;

(ii) expressing said rare-cutting endonuclease in the cell together with a polynucleotide encoding a fusion protein that comprises:
a first polypeptide that is a human TREX2 exonuclease protomer of SEQ ID NO: 26 or functional mutant thereof that shares at
least 80% identity with the human TREX2 exonuclease protomer;

a second polypeptide that is a human TREX2 exonuclease protomer of SEQ ID NO: 26 or functional mutant thereof that shares
at least 80% identity with the human TREX2 exonuclease protomer; and

a peptidic linker connecting said first and second polypeptides;
(iii) thereby obtaining, by expression of said fusion protein in said cell, increased double strand-break-induced mutagenesis
at said genomic locus of interest.

US Pat. No. 9,290,748

USE OF ENDONUCLEASES FOR INSERTING TRANSGENES INTO SAFE HARBOR LOCI

Cellectis, Paris (FR)

1. A method for inserting a transgene into the genome of a human cell comprising:
a) selecting, within the genome of said human cell, a retroviral insertion site (RIS) that is neither associated with cancer
nor with abnormal cell proliferation;

b) bringing said human cell in contact with an endonuclease capable of cleaving a target sequence of 12-45 base pairs that
is located at a distance of at most 200 kb from the RIS,

wherein the endonuclease comprises a sequence selected from the group consisting of SEQ ID Nos. 81, 82-85, 294, 295, 76, 77,
79-80, 25-40, 86-96, 127-150, 182-213, 235, 236, 238, 239, 241, 242, 244, 245, 250-252, 254-270, and 275-278;

c) bringing said human cell contacted according to step b) in contact with a targeting construct comprising a transgene and
two sequences homologous to the genomic sequence flanking the target sequence, thereby inserting said transgene into said
genome,

wherein said target sequence is located within a locus selected from the group consisting of the SH6 locus on human chromosome
21 at 21q21.1, the SH3 locus on human chromosome 6 at 6p25.1, the SH4 locus on human chromosome 7 at 7q31.2, the SH12 locus
on human chromosome 13 at 13q34, the SH13 locus on human chromosome 3 at 3p12.2, the SH19 locus on human chromosome 22, the
SH20 locus on human chromosome 12 at 12q21.2, the SH21 locus on human chromosome 3 at 3p24.1, the SH33 locus on human chromosome
6 at 6p12.2, the SH7 locus on human chromosome 2 at 2p16.1, the SH8 locus on human chromosome 5, the SH18 locus, the SH31
locus, the SH38 locus, the SH39 locus, the SH41 locus, the SH42 locus, the SH43 locus, the SH44 locus, the SH45 locus, the
SH46 locus, the SH47 locus, the SH48 locus, the SH49 locus, the SH50 locus, the SH51 locus, the SH52 locus, the SH70 locus,
the SH71 locus, the SH72 locus, the SH73 locus, the SH74 locus, the SH75 locus, the SH101 locus, the SH106 locus, the SH107
locus, the SH102 locus, the SH105 locus, the SH103 locus, the SH104 locus, the SH113 locus, the SH109 locus, the SH112 locus,
the SH108 locus, the SH110 locus, the SH114 locus, the SH116 locus, the SH111 locus, the SH115 locus, the SH121 locus, the
SH120 locus, the SH122 locus, the SH117 locus, the SH118 locus, the SH119 locus, the SH123 locus, the SH126 locus, the SH128
locus, the SH129 locus, the SH124 locus, the SH131 locus, the SH125 locus, the SH127 locus, the SH130 locus , the SH11 locus,
the SH17 locus, the SH23 locus, the SH34 locus, the SH40 locus, the SH53 locus, the SH54 locus, the SH55 locus, the SH56 locus,
the SH57 locus, the SH58 locus, the SH59 locus, the SH60 locus, the SH61 locus, the SH62 locus, the SH65 locus, the SH67 locus,
the SH68 locus and the SH69 locus.

US Pat. No. 9,944,709

CD123 SPECIFIC CHIMERIC ANTIGEN RECEPTORS FOR CANCER IMMUNOTHERAPY

CELLECTIS, Paris (FR)

1. A method of treating a pre-malignant cancer, a malignant cancer, or a relapse refractory cancer expressing CD123, comprising administering to a patient with a CD123 expressing pre-malignant cancer, malignant cancer or a CD123 expressing relapse refractory cancer a therapeutically effective amount of a pharmaceutical composition comprising an engineered immune cell expressing a CD123 specific Chimeric Antigen Receptor (CAR) comprisingan extracellular ligand binding-domain comprising a heavy chain variable region (VH) and a light chain variable region (VL) from a monoclonal anti-CD123 antibody, wherein the extracellular ligand domain comprises CDR sequences of SEQ ID NOs: 67, 68, and 69 and SEQ ID NOs: 70, 71, and 72,
a Fc?RIII?, CD8?, or IgG1 hinge,
a CD8? or 4-1BB transmembrane domain, and
a cytoplasmic domain comprising a CD3-? signaling domain and a co-stimulatory domain from 4-1BB.
US Pat. No. 9,365,864

MEGANUCLEASE RECOMBINATION SYSTEM

CELLECTIS, Paris (FR)

1. A method for transforming by homologous recombination at least one cell in vitro, comprising:
(a) stably transforming at least one cell by inserting construct (i), which is encoded by a nucleic acid molecule, which comprises:
A1-A2-A3-A4-A5  (i),
wherein A1 is a first promoter, A2 is a first homologous portion, A3 is a meganuclease cleavage site, A4 is a first marker
gene, A5 consists of the neomycin resistance gene of SEQ ID NO:7, and wherein construct (i) is configured to be stably integrated
into the genome of at least one target cell, into the genome of said at least one cell;

(b) cloning a sequence coding for a gene of interest into position B3 of construct (ii), which is encoded by a nucleic acid
molecule, which comprises at least the following components:

A2? -B1-B2-B3-B4-A5?  (ii),
wherein A2? comprises a portion of said first homologous portion A2, B1 is a second marker gene different from said first
marker gene, B2 is a second promoter, B3 is a multiple cloning site, B4 is a third promoter, A5? consists of the inactive
neomycin resistance gene of SEQ ID NO:13;

(c) co-transfecting said cell of (a) with said construct (ii) of (b) and construct (iii), (iv), or (v), which are encoded
by nucleic acid molecules, which comprise components:

C1-C2  (iii),
C3  (iv), or
an isolated or recombinant protein which comprises component:
C4  (v),
wherein C1 is a fourth promoter, C2 is the open reading frame (ORF) of a meganuclease, C3 is a messenger RNA (mRNA) encoding
said meganuclease, and C4 is an isolated or recombinant protein of said meganuclease,

wherein a meganuclease from construct (iii), (iv), or (v) recognizes and cleaves A3, and construct (iii), (iv), or (v) are
configured to be co-transfected with construct (ii) into said at least one target cell;

(d) following homologous recombination between said construct (ii) and said stably inserted construct (i), selecting at least
one cell from (c) based upon: the absence of a first marker gene encoded by component A4 of said construct (i) and the activity
of a second marker gene encoded by component B1 and neomycin resistance activity.

US Pat. No. 10,000,746

LAGLIDADG HOMING ENDONUCLEASE CLEAVING THE T CELL RECEPTOR ALPHA GENE AND USES THEREOF

CELLECTIS, Paris (FR) PR...

1. A polypeptide comprising an I-Onul variant having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10,wherein said I-Onul variant cleaves a target nucleic acid sequence within the T cell receptor alpha constant gene (TRAC),
wherein said I-Onul variant is fused to a TALE DNA binding domain.
US Pat. No. 10,113,162

MODIFYING SOYBEAN OIL COMPOSITION THROUGH TARGETED KNOCKOUT OF THE FAD2-1A/1B GENES

CELLECTIS, Paris (FR)

1. A soybean plant, plant part, or plant cell comprising:(a) a deletion in each FAD2-1A allele, wherein said deletion in each FAD2-1A allele was induced by transcription activator-like (TAL) effector endonucleases targeted to SEQ ID NOS: 32 and 33, and
(b) a deletion in each FAD2-1B allele, wherein said deletion in each FAD2-1B allele is 23 bp in size, and wherein said deletion in each FAD2-1B allele was induced by TAL effector endonucleases targeted to SEQ ID NOS: 32 and 33,
wherein oil produced from said plant, plant part, or plant cell has increased oleic acid content and decreased linoleic acid content as compared to oil produced from a corresponding wild type soybean plant, plant part, or plant cell.
US Pat. No. 9,890,393

METHODS FOR ENGINEERING T CELLS FOR IMMUNOTHERAPY BY USING RNA-GUIDED CAS NUCLEASE SYSTEM

CELLECTIS, Paris (FR)

1. A method of preparing T-cells for immunotherapy comprising the step of:
(a) genetically modifying primary T-cells by introduction and/or expression into the cells of at least:
a RNA-guided endonuclease; and
a specific guide RNA that directs said endonuclease to at least one targeted locus in the T-cell genome,
wherein said RNA-guided endonuclease is expressed from transfected mRNA;
wherein said RNA-guided endonuclease comprises the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2; and
(b) expanding the resulting cells.
US Pat. No. 10,508,289

RARE-CUTTING ENDONUCLEASES FOR EFFICIENT AND SPECIFIC TARGETING DNA SEQUENCES COMPRISING HIGHLY REPETITIVE MOTIVES

CELLECTIS, Paris (FR)

1. A method of contracting a repeat sequence within a genetic sequence in a cell, comprising:(a) selecting a target sequence spanning the repeat sequence and a region adjacent to the repeat sequence;
(b) providing a rare-cutting endonuclease capable of binding said target sequence and cleaving the repeat sequence; and
(c) introducing said rare-cutting endonuclease into said cell,
wherein said rare-cutting endonuclease binds to the target sequence by recognizing at least 10 nucleic acid bases adjacent to the repeat sequence and at least 5 nucleic acid bases inside the repeat sequence;
such that the rare-cutting endonuclease induces cleavage within repeat sequence and induces contraction of the repeat sequence by Single Stranded Annealing, wherein the rare-cutting endonuclease has at least 80% amino acid sequence identity with a sequence selected from the group consisting of SEQ ID NO: 8, 10 and 15.
US Pat. No. 10,544,201

ROR1 SPECIFIC MULTI-CHAIN CHIMERIC ANTIGEN RECEPTOR

CELLECTIS, Paris (FR)

1. A ROR1 specific multi-chain Chimeric Antigen Receptor (mcCAR) comprising:(a) a transmembrane polypeptide from the alpha chain of high-affinity IgE receptor (Fc?RI) fused to an extracellular ROR1 ligand binding domain;
(b) a second transmembrane polypeptide from the gamma chain of Fc?RI fused to a signal transducing domain of CD3zeta; and
(c) a third transmembrane polypeptide from the beta chain of Fc?RI comprising a costimulatory domain of 4-1BB,
wherein said ROR1 ligand binding domain fused to said alpha chain of Fc?RI is a single-chain variable fragment (scFv) comprising variable heavy (VH) and variable light (VL) chains conferring specificity to ROR1, wherein:
(i) said VH chain comprises a polypeptide sequence displaying at least 90% identity to SEQ ID NO: 28, and said VL chain comprises a polypeptide displaying at least 90% sequence identity to SEQ ID NO:32, wherein said VH chain comprises SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31, and wherein said VL chain comprises SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35;
(ii) said VH chain comprises a polypeptide sequence displaying at least 90% identity to SEQ ID NO: 12, and said VL chain comprises a polypeptide displaying at least 90% sequence identity to SEQ ID NO:16, wherein said VH chain comprises SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, and wherein said VL chain comprises SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19;
(iii) said VH chain comprises a polypeptide sequence displaying at least 90% identity to SEQ ID NO: 20, and said VL chain comprises a polypeptide displaying at least 90% sequence identity to SEQ ID NO:24, wherein said VH chain comprises SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, and wherein said VL chain comprises SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27;
(iv) said VH chain comprises a polypeptide sequence displaying at least 90% identity to SEQ ID NO: 36, and said VL chain comprises a polypeptide displaying at least 90% sequence identity to SEQ ID NO:40, wherein said VH chain comprises SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, and wherein said VL chain comprises SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43;
(v) said VH chain comprises a polypeptide sequence displaying at least 90% identity to SEQ ID NO:44, and said VL chain comprises a polypeptide displaying at least 90% sequence identity to SEQ ID NO:48, wherein said VH chain comprises SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47, and wherein said VL chain comprises SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51;
(vi) said VH chain comprises a polypeptide sequence displaying at least 90% identity to SEQ ID NO: 52, and said VL chain comprises a polypeptide displaying at least 90% sequence identity to SEQ ID NO:56, wherein said VH chain comprises SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55, and wherein said VL chain comprises SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:59;
(vii) said VH chain comprises a polypeptide sequence displaying at least 90% identity to SEQ ID NO: 60, and said VL chain comprises a polypeptide displaying at least 90% sequence identity to SEQ ID NO:64, wherein said VH chain comprises SEQ ID NO:61, SEQ ID NO:62, and SEQ ID NO:63, and wherein said VL chain comprises SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67; or
(viii) said VH chain comprises a polypeptide sequence displaying at least 90% identity to SEQ ID NO:68, and said VL chain comprises a polypeptide displaying at least 90% sequence identity to SEQ ID NO:72, wherein said VH chain comprises SEQ ID NO:69, SEQ ID NO:70, and SEQ ID NO:71, and wherein said VL chain comprises SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75.

US Pat. No. 10,494,626

DYNAMIC MIXING AND ELECTROPORATION CHAMBER AND SYSTEM

CELLECTIS S.A., (FR)

1. An electroporation apparatus comprising:a mixing chamber comprising two opposite walls, one opposite wall serving as a first electrode and the other opposite wall serving as a second electrode;
a space between the two opposite walls bounded by remaining walls of the mixing chamber;
wherein the remaining walls of the chamber comprises a top chamber wall section, a first side chamber wall section, a bottom chamber wall section, and a second side chamber wall section;
wherein the first side chamber wall section is adjacent and perpendicular to the top chamber wall section and further comprises a first curved portion connecting to the first side chamber wall section to the bottom chamber wall section;
wherein the second side chamber wall section is present opposite to the first side chamber wall section and further comprises a second curved portion connecting the second side chamber wall section to the top chamber wall section;
at least two inlet ports oriented nonparallel and adjacent to each other and present at a top corner of the mixing chamber, wherein the top corner is a corner of the mixing chamber comprising the top chamber wall section and the first side chamber wall section; and
an outlet port in communication with the second curved portion, and
wherein the mixing chamber is configured to mix liquids entering the mixing chamber throw the at least two inlet ports gently and thoroughly.
US Pat. No. 10,342,829

MULTI-CHAIN CHIMERIC ANTIGEN RECEPTOR AND USES THEREOF

CELLECTIS, Paris (FR)

1. A multi-chain Chimeric Antigen Receptor (CAR) including at least two different and separate transmembrane polypeptides expressed on an immune cell, comprising:a first transmembrane polypeptide fused with an extracellular ligand-binding domain, the extracellular ligand-binding domain comprising a single-chain antibody fragment (scFv) that recognizes an antigen on a target cell, wherein the first transmembrane polypeptide comprises an alpha chain of Fc epsilon Receptor I (Fc?RI) without its high affinity IgE binding domain, and
at least a second transmembrane polypeptide comprising at least one intracellular signal transducing domain from a T cell Receptor (TCR) zeta chain;
wherein the first transmembrane polypeptide dimerizes with the second transmembrane polypeptide to form the multi-chain CAR on the immune cell.
US Pat. No. 10,286,007

USE OF PRE T ALPHA OR FUNCTIONAL VARIANT THEREOF FOR EXPANDING TCR ALPHA DEFICIENT T CELLS

CELLECTIS, Paris (FR)

1. A method to obtain modified TCR alpha deficient human T-cells, wherein said TCR alpha deficient human T-cells can proliferate independently of the Major Histocompatibility Complex signaling pathway, said method comprising the following steps:(a) recovering cells from a human;
(b) genetically modifying said recovered cells ex-vivo by inactivating TCR alpha;
(c) introducing into said genetically modified cells an exogenous pTalpha polypeptide that supports CD3 surface expression; and
(d) expanding TCR alpha deficient human T-cells.
US Pat. No. 10,087,453

MODIFIED DIATOMS FOR BIOFUEL PRODUCTION

CELLECTIS, Paris (FR)

1. A diatom strain, which has been genetically modified in a gene involved in lipid metabolism selected from the group consisting of: delta-12 desaturase, omega-3 desaturase, palmitoyl protein thioesterase and Enoyl ACP reductase, wherein said gene has been inactivated, and wherein said diatom strain produces an increased amount, storage and/or quality of lipids in comparison with its wild type, and has a lipid content of at least 40% of its dry weight.
US Pat. No. 10,472,613

METHOD FOR MODULATING CAR-INDUCED IMMUNE CELLS ACTIVITY

CELLECTIS, Paris (FR)

1. A method comprising:contacting an engineered immune cell with an external ligand,
wherein the engineered immune cell co-expresses a Chimeric Antigen Receptor (CAR) comprising a first dimerization domain comprising FKBP or FRB, the CAR being activated in vivo and/or in vitro by the external ligand, and an engineered inhibitory membrane protein (IMP) complex, the IMP comprising at least one intracellular inhibitory signaling domain from PD1 and one second dimerization domain comprising FKBP or FRB, and
wherein the external ligand binds to the CAR, which results in a signal transduction through the CAR; and
adding rapamycin or a rapalog that binds to the first dimerization domain comprising FKBP or FRB of the CAR and to the second dimerization domain comprising FKBP or FRB of the IMP to form a non-covalently bonded macromolecular complex between said CAR and IMP.
US Pat. No. 10,426,795

USE OF PRETALPHA OR FUNCTIONAL VARIANT THEREOF FOR EXPANDING TCRALPHA DEFICIENT T CELLS

CELLECTIS, Paris (FR)

1. An isolated TCRalpha deficient primary human T cell,wherein the TCRalpha gene in the T cell has been inactivated by introducing a rare-cutting endonuclease that induces a DNA cleavage within the TCRalpha gene,
comprising an exogenous nucleic acid encoding an exogenous pTalpha polypeptide that supports CD3 surface expression and supports expansion of the TCR-deficient primary T cell using stimulatory anti-CD3/CD28 antibodies.
US Pat. No. 10,378,026

RNA BASED METHOD TO OBTAIN STABLY INTEGRATED RETROVIRAL VECTORS

CELLECTIS, Paris (FR)

1. A method comprising:(a) providing at least one RNA comprising: (i) a sequence encoding foamy virus Gag protein, (ii) a sequence encoding foamy virus Pol protein; (iii) a Foamy Virus genomic sequence and a transgene; and
(b) introducing said RNA into a cell by electroporation, such that the transgene is integrated within the genome of the electroporated cell.
US Pat. No. 10,363,270

METHODS FOR ENGINEERING ALLOGENEIC AND IMMUNOSUPPRESSIVE RESISTANT T CELL IMMUNOTHERAPY

CELLECTIS, Paris (FR)

1. A method for treating a cancer patient using immunotherapy comprising:providing primary human T cells from a single donor;
co-electroporating into said primary human T-cells:
(a) RNAs encoding two half TALE-nucleases having Fok-I catalytic domains, wherein the two half TALE-nucleases dimerize and recognize two half-targets separated by a spacer of 11-15 bp and cleave the target sequence of SEQ ID NO:40 within a gene encoding CD52, and
(b) RNAs encoding two half TALE-nucleases having Fok-I catalytic domains,
wherein the two half TALE-nucleases dimerize and recognize two half-targets separated by a spacer of 11-15 bp and cleave the target sequence of SEQ ID NO:37 within a gene encoding TCR?,
to generate doubly-inactivated human T-cells having both the CD52 and the TCR? genes inactivated;
modifying the doubly inactivated human T-cells to express a chimeric antigen receptor; and
infusing at least 104 of the doubly inactivated human T-cells expressing a chimeric antigen receptor into said cancer patient for immunotherapy treatment.

US Pat. No. 10,239,948

METHOD OF ENGINEERING MULTI-INPUT SIGNAL SENSITIVE T CELL FOR IMMUNOTHERAPY

CELLECTIS, Paris (FR)

1. A method for treating cancer in a patient in need thereof comprising administering to said patient an immune cell comprising a multi-chain chimeric antigen receptor (CAR) comprising:an extracellular ligand binding domain capable of recognizing a specific ligand expressed at the surface of a tumor cell, said extracellular ligand binding domain further comprising a hinge selected from CD8?, CD4, CD28, RTK, IgGI, and EpoR2-D2;
a transmembrane domain;
an intracellular domain comprising a CD3? domain; and
an oxygen-sensitive polypeptide domain,
wherein said multi-chain CAR comprises part of the alpha, beta, and gamma chains from an Fc receptor comprising an amino acid sequence identity greater than 80% to SEQ ID NO: 7, 3 and 4, respectively.
US Pat. No. 10,196,608

METHOD FOR IN SITU INHIBITION OF REGULATORY T CELLS

CELLECTIS, Paris (FR)

1. A method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an engineered T-cell comprising:a) a first exogenous nucleic acid molecule comprising a nucleotide sequence coding for a first chimeric antigen receptor (CAR) comprising an extracellular domain capable of binding to at least one antigen expressed at the surface of a malignant cell; and
b) at least a second exogenous nucleic acid molecule comprising a nucleotide sequence coding for a peptide-inhibitor of FoxP3 comprising the amino acid sequence of SEQ ID NO 1 or SEQ ID NO:3,
wherein the peptide-inhibitor of FoxP3 inhibits regulatory T-cell activity.
US Pat. No. 10,550,402

MODIFYING SOYBEAN OIL COMPOSITION THROUGH TARGETED KNOCKOUT OF THE FAD3A/B/C GENES

CELLECTIS, Paris (FR)

1. A soybean plant, plant part, or plant cell comprising deletions in one or more FAD3A alleles, one or more FAD2-1A alleles, and one or more FAD2-1B alleles, wherein said deletions were induced by TAL effector endonucleases targeted specifically to FAD3A alleles, FAD2-1A alleles, and FAD2-1B alleles, wherein said TAL effector endonucleases comprise a TAL effector endonuclease targeted to said one or more FAD3A alleles that binds to the sequences as set forth in SEQ ID NOs: 8 and 9, and a TAL effector endonuclease targeted to said one or more FAD2-1A alleles and FAD2-1B alleles that binds to the sequences as set forth in SEQ ID NOs: 41 and 42, and wherein said deletions in said one or more FAD3A alleles comprise a 4 bp size deletion and the 43 bp size deletion of SEQ ID NO: 24 as set forth in SEQ ID NO: 104; wherein said deletions in said one or more FAD2-1A alleles are 63 bp in size, and said deletions in said one or more FAD2-1B alleles are 23 bp in size; and wherein the soybean plant, plant part, or plant cell produces oil that has less than 4.3% linolenic acid content, increased oleic acid content, and decreased linoleic acid content as compared to oil produced from a corresponding wild type soybean plant, plant part, or plant cell.

US Pat. No. 10,472,396

MODULAR BASE-SPECIFIC NUCLEIC ACID BINDING DOMAINS FROM BURKHOLDERIA RHIZOXINICA PROTEINS

CELLECTIS, Paris (FR)

1. A nucleic acid binding polypeptide that comprises a succession of at least 12 modules, wherein each module consists of 30 to 35 contiguous amino acid residues, wherein said succession of modules displays a base per base specificity towards a nucleic acid target sequence,wherein at least one of said modules has at least 70% sequence identity with one of the module polypeptide sequences from proteins E5AV36, E5AW43 and E5AW45 of Burkholderia rhizoxinica;
wherein said nucleic acid binding polypeptide is fused to a catalytic domain from I-Tevl, and
wherein said polypeptide binds to and cleaves the nucleic acid target sequence on a chromosome in a mammalian cell.
US Pat. No. 10,378,007

METHODS FOR MODULATING TAL SPECIFICITY

CELLECTIS, Paris (FR)

1. A method for synthesizing a polynucleotide sequence encoding a transcription activator-like effector (TALE) protein, said protein having alternative targeting specificity towards a nucleic acid target sequence containing a nucleic acid base(s) G, wherein said method comprises assembling at least 9 polynucleotide sequences encoding RVDs (repeat variable disresidue), each RVD having specificity to a nucleic acid base in said nucleic acid target, and wherein at least one of said 9 RVDs are selected among the alternative RVD code:NI or NG to target G, andwherein the RVD is within an RVD triplet selected from: NI-HD-NI for ATG; NI-HD-NI for GCA; NI-HD-NI for GTA; NI-HD-NN for GCG; NI-NN-NI for GAA; NI-NN-HD for GGC; NI-NN-NN for GAG; NI-NN-NN for GGG; NI-NN-NG for AGG; NI-NN-NG for GGT; NI-NG-NI for ATG; NI-NG-NI for GTA; NI-NG-HD for AGC; NI-NG-HD for GAC; NI-NG-HD for GTA; NI-NG-HD for GTC; NI-NG-NN for AGG; NI-NG-NN for GTA; NI-NG-NN for GTG; NI-NG-NG for GTT; HD-NN-NI for AAG; HD-NN-NI for AGG; HD-NN-NI for CGG; HD-NN-NG for AGG; HD-NN-NG for CGG; HD-NG-NI for ATG; HD-NG-NI for CGA; HD-NG-NI for CTG; HD-NG-HD for AGC; HD-NG-NN for AGG; HD-NG-NN for CGG; HD-NG-NG for AGT; HD-NG-NG for ATG; HD-NG-NG for CGT; HD-NG-NG for CTG; NN-NI-NI for AGA; NN-NI-NI for GGA; NN-NI-HD for AGC; NN-NI-HD for GGC; NN-NI-NN for GGA; NN-NI-NG for AGT; NN-NI-NG for GAG; NN-NI-NG for GGT; NN-HD-NI for AAG; NN-HD-NI for ACG; NN-HD-NI for GCG; NN-NN-NI for AAG; NN-NN-NI for AGG; NN-NN-NI for GAG; NN-NN-NI for GGG; NN-NN-NG for AGG; NN-NN-NG for GAG; NN-NN-NG for GGG; NN-NG-NI for AGA; NN-NG-NI for ATG; NN-NG-NI for GTG; NN-NG-HD for AGC; NN-NG-NN for AGG; NN-NG-NN for GGG; NN-NG-NG for ATG; NN-NG-NG for GGT; NN-NG-NG for GTG; NG-NI-NI for GAA; NG-NI-NI for TGA; NG-NI-HD for GAC; NG-NI-HD for TGC; NG-NI-NN for GAG; NG-NI-NN for TGA; NG-NI-NG for AGT; NG-NI-NG for GAT; NG-NI-NG for TAG; NG-NI-NG for TGT; NG-HD-NI for GCA; NG-HD-NI for TCG; NG-HD-HD for GCC; NG-HD-NN for GCA; NG-HD-NN for GCG; NG-NN-NI for GAA; NG-NN-NI for GGA; NG-NN-NI for TGG; NG-NN-HD for GAC; NG-NN-HD for GCC; NG-NN-HD for GGC; NG-NN-NN for GAG; NG-NN-NN for GGA; NG-NN-NN for GGG; NG-NN-NG for GGT; NG-NN-NG for TAG; NG-NN-NG for TGG; NG-NG-HD for GTC; NG-NG-HD for TGC; NG-NG-NN for GTA; NG-NG-NN for GTG; NG-NG-NN for TGG; NI-HD-NI for ATG; NI-HD-NI for GTA; NI-NG-HD for GAC; NG-NI-NG for AGT; and NG-NN-HD for GCC.
US Pat. No. 10,316,101

BCMA (CD269) SPECIFIC CHIMERIC ANTIGEN RECEPTORS FOR CANCER IMMUNOTHERAPY

CELLECTIS, Paris (FR)

1. A method for treating a pathological condition related to a B-cell maturation antigen (BCMA)-expressing cancer cell in a patient in need thereof, the method comprising:administering to the patient a therapeutically effective amount of a composition comprising an engineered T cell comprising a cell surface-expressed BCMA specific Chimeric Antigen Receptor (CAR), wherein the BCMA specific CAR comprises:
(a) an extracellular ligand binding-domain comprising a VH and a VL from a monoclonal anti-BCMA antibody, wherein said VH comprises SEQ ID NO. 68 and said VL comprises SEQ ID NO. 69; said VH comprises SEQ ID NO. 11 and said VL comprises SEQ ID NO. 12; or said VH comprises SEQ ID NO. 13 and said VL comprises SEQ ID NO. 14;
(b) a Fc?RIII? hinge, a CD8? hinge or an IgG1 hinge comprising a sequence having at least 90% sequence identity with SEQ ID NOs. 3, 4, and 5, respectively;
(c) a CD8? transmembrane domain; and
(d) an intracellular cytoplasmic domain comprising a CD3 zeta signaling domain and a co-stimulatory domain from 4-1BB;
wherein said BCMA specific CAR-expressing T cell treats the pathological condition related to the BCMA-expressing cancer cell by inducing cytolytic activity in the cancer cell in the patient.
US Pat. No. 10,301,614

METHOD TO OVERCOME DNA CHEMICAL MODIFICATIONS SENSITIVITY OF ENGINEERED TALE DNA BINDING DOMAINS

CELLECTIS, Paris (FR)

1. A method to process a nucleic acid target sequence comprising a 5-methyl-cytosine comprising:(a) providing cells containing a nucleic acid target sequence that comprises a 5-methyl-cytosine;
(b) introducing into said cell a polynucleotide comprising:
(i) a first polynucleotide encoding a transcription activator-like effector (TALE) protein comprising a plurality of TALE-like repeat sequences, each repeat comprising a repeat variable-diresidue (RVD) specific to each nucleic acid base of said nucleic acid target sequence, wherein the RVD that specifically targets the 5-methyl-cytosine within said nucleic acid target sequence is selected from N*, T*, Q* and H*, wherein * represents a gap in one position of the RVD; and
(ii) a second polynucleotide encoding an additional protein domain that has a nuclease activity, polymerase activity, kinase activity, phosphatase activity, methylase activity, topoisomerase activity, integrase activity, transposase activity, ligase activity, helicase activity or a recombinase activity;
(c) expressing said polynucleotide to form a chimeric protein that binds said nucleic acid target sequence and processes the nucleic acid within or adjacent to said nucleic acid target sequence and,
(d) selecting the cells in which said TALE protein has processed said nucleic acid target sequence within or adjacent to said nucleic acid target sequence;
wherein the TALE protein comprising an RVD N*, T*, Q* or H* that specifically targets the 5-methyl-cytosine can bind said nucleic acid target sequence more efficiently than a variant TALE protein having the RVD NG at the same position.
US Pat. No. 10,301,637

POTATOES WITH REDUCED GRANULE-BOUND STARCH SYNTHASE

CELLECTIS, Paris (FR)

1. A method for making a Solanum plant, comprising:(a) contacting a population of Solanum plant cells comprising a functional GBSS allele with a rare-cutting endonuclease targeted to an endogenous GBSS sequence, wherein said rare-cutting endonuclease is a TALE-nuclease, and wherein said TALE-nuclease binds to a sequence as set forth in SEQ ID NO:2 or SEQ ID NO:3,
(b) selecting, from said population, a cell in which at least three GBSS alleles have been inactivated, wherein each of said at least three GBSS alleles comprises a deletion of more than one nucleotide base pair, and
(c) growing said selected plant cell into a Solanum plant, wherein said Solanum plant has reduced levels of amylose as compared to a control Solanum plant in which said at least three GBSS alleles have not been inactivated.
US Pat. No. 10,584,352

METHODS FOR ENGINEERING T CELLS FOR IMMUNOTHERAPY BY USING RNA-GUIDED CAS NUCLEASE SYSTEM

CELLECTIS, Paris (FR)

1. A method of preparing and administering T-cells for immunotherapy comprising the steps of:(a) providing primary human T-cells from a donor,
(b) genetically modifying the primary human T-cells to eliminate expression of the T-cell receptor (TCR), comprising expressing in the cells
(i) a Cas9 endonuclease fused to a nuclear localization signal (NLS), and
(ii) a guide RNA that directs said endonuclease to at least one targeted locus encoding the TCR in the T-cell genome,
(c) expanding the genetically modified T-cells, and
(d) administering at least 104 of the expanded genetically modified T-cells to a patient.
US Pat. No. 10,526,406

CHIMERIC ANTIGEN RECEPTOR USING ANTIGEN RECOGNITION DOMAINS DERIVED FROM CARTILAGINOUS FISH

CELLECTIS, Paris (FR)

1. A chimeric antigen receptor (CAR) comprising:one extracellular antigen recognition domain comprising at least one Complementary Determining Region (CDR) of a variable new antigen receptor (VNAR) polypeptide,
wherein the CAR is a multi-chain CAR, comprising a first transmembrane polypeptide and a second transmembrane polypeptide that are separate and different transmembrane polypeptides expressed on an immune cell,
wherein the first transmembrane polypeptide is fused with the extracellular antigen recognition domain comprising at least one CDR that recognizes an antigen on a target cell and wherein the first transmembrane polypeptide does not contain a signal transducing domain,
and wherein the first transmembrane polypeptide does not contain a signal transducing domain, and wherein the first transmembrane polypeptide does not contain a co-stimulatory domain, and
wherein the first and the second transmembrane polypeptides multimerize to form a multi-chain CAR.
US Pat. No. 10,517,896

USE OF PRE T ALPHA OR FUNCTIONAL VARIANT THEREOF FOR EXPANDING TCR ALPHA DEFICIENT T CELLS

CELLECTIS, Paris (FR)

1. A combination therapy comprising:(a) administering an engineered T-cell comprising an inactivated CD52 gene; and
(b) administering an anti-CD52 antibody.
US Pat. No. 10,513,698

POTATOES WITH REDUCED COLD-INDUCED SWEETENING

CELLECTIS, Paris (FR)

1. A Solanum tuberosum plant, plant part, or plant cell comprising a deletion in at least two vacuolar invertase (VInv) alleles endogenous to said plant, plant part, or plant cell, wherein said deletion was induced by introducing one or more rare-cutting endonucleases into a S. tuberosum cell, such that said plant, plant part, or plant cell has reduced expression of VInv as compared to a control S. tuberosum plant, plant part, or plant cell that lacks said deletion, and wherein said deletion is at a target sequence as set forth in SEQ ID NO:1, or at a target sequence having at least 95% identity to SEQ ID NO:1, and wherein each of said at least two VInv alleles having a deletion comprises the sequence set forth in SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34.
US Pat. No. 10,357,515

METHOD FOR GENERATING BATCHES OF ALLOGENEIC T-CELLS WITH AVERAGED POTENCY

CELLECTIS, Paris (FR)

1. A method for generating a batch of T-cells originating from different human donors, said method comprising the steps of:(a) obtaining T-cell samples from individual human donors;
(b) introducing a rare-cutting endonuclease disrupting at least one gene encoding a T cell Receptor (TCR) component into the cells of each individual sample;
(c) purifying the TCR negative cells from the sample; and
(d) pooling the TCR negative cells originating from the samples from 3 to 50 individual donors; to obtain a batch of T-cells to be used off-the-shelf.