WO2023014839A1 - Methods for the treatment of sickle cell disease - Google Patents

Abstract

Disclosed herein are ex vivo methods and compositions for treating sickle cell disease.

Description

METHODS FOR THE TREATMENT OF SICKLE CELL DISEASE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Patent Application No. 63/228,988, filed August 3, 2021 , and No. 63/280,482, filed November 17, 2021 . The disclosures of both priority applications are incorporated by reference herein in their entirety.

BACKGROUND

[0002] Sickle cell disease (SCD) is a genetic hematologic disorder affecting an estimated 70,000 to 100,000 individuals in the United States. SCD is caused by a mutation in the p- globin gene (Glu6Val) that results in the abnormal hemoglobin, sickle hemoglobin (HbS). When inherited on both alleles, the sickle mutation produces the homozygous genotype HbSS. This genotype, occurring in >70% of cases worldwide, gives rise to the most severe form of the disease. Saraf et a/. (2014) Paediatr Respir Rev. 15(1):4-12. The sickle mutation can also be inherited in trans with an allele specifying a dysfunctional p-globin (genotype HbS-p[+]- thalassemia), an absent p-globin (genotype HbS-p°-thalassemia), or a different p-globin point mutation, such as hemoglobin C (leading to HbSC disease). HbS-p°-thalassemia is clinically indistinguishable from genotype HbSS, while the other 2 genotypes are generally less severe in phenotype.

[0003] For the majority of patients with SCD, treatment focuses on supportive care, pain management, and the surgical or medical management of end organ damage. Hydoxyurea (HU) treatment reduces the likelihood of patient pain crisis that requires hospitalization and prevents acute chest syndrome (ACS). Recently, two compounds have been approved in the US for preventing vaso-occlusive crisis (VOC) and to mitigate anemia . Transfusion therapy is used in SCD in the context of some acute complications, such as stroke, ACS, splenic sequestration, or aplastic crisis, or chronically for severe anemia, stroke, and pain crisis prevention. Despite its benefits, use of chronic red blood cell (RBC) transfusion leads to iron overload in the heart and liver (and the need for chelation therapy) and increases the risks of alloimmunization, autoantibody formation, and blood-borne infection.

[0004] The only curative therapy currently available for SCD is a hematopoietic stem cell transplant (HSCT) from an immunologically matched donor. However, only about 20% of SCD patients are estimated to have a matched donor available. Hsieh et al. (2011). Blood. 118(5): 1197-1207. There remains a need for compositions and methods for treating and/or preventing SCD. SUMMARY OF THE DISCLOSURE

[0005] From the description herein, it will be appreciated that that the present disclosure encompasses multiple aspects and embodiments which include, but are not limited to, the following:

[0006] In one aspect, disclosed herein is an ex vivo method of treating sickle cell disease (SCD) in a subject, the method comprising: administering to the subject in need thereof a composition comprising a genetically modified cell comprising a red blood cell (RBC) precursor cell, wherein the precursor cell comprises SB-mRENH1 mRNAs and SB-mRENH2 mRNAs, which encode a ZFN pair; and a genomic modification made following cleavage by the ZFN pair, wherein the modification is within an endogenous BCL11A enhancer sequence. In some embodiments, the composition further comprises a cell descended from the genetically modified cell comprising the SB-mRENH1 mRNAs and the SB-mRENH2 mRNAs.

[0007] In some embodiments of the methods disclosed herein, the sickle cell disease is severe sickle cell disease. In some embodiments of the methods disclosed herein, the subject has adequate physiological function by BMT CNT guidelines prior to administering of the composition.

[0008] In some embodiments of the methods disclosed herein, the fetal hemoglobin (HbF) production in the subject (i.e. with SCD) is increased after administration of the disclosed composition comprising the genetically modified cells. In some embodiments of the methods disclosed herein, one or more clinical symptoms of sickle cell disease in the subject are decreased, ameliorated, or eliminated. In some embodiments of the methods disclosed herein, one of the clinical symptoms in the subject that is decreased, ameliorated, or eliminated is vaso-occlusive crisis (VOC). In some embodiments of the methods disclosed herein, a change from baseline of clinical laboratory hemoglobin fractions in grams/dL plasma and/or percent HbF of total hemoglobin (Hb) is achieved in the subject. In some embodiments, the effect of the composition on the subject is realized within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more days after administration of the genetically modified cells.

[0009] In some embodiments of the methods disclosed herein, the levels of SCD-related disease biomarkers in the subject are altered following treatment. In some embodiments, the SCD-related disease biomarkers are changes in iron metabolism and/or changes in levels of erythropoietin, haptoglobin, and/or hepcidin.

[0010] In some embodiments of the methods disclosed herein, the clinical symptoms associated with iron overload or associated with baseline transfusion therapy in the subject are ameliorated or eliminated in the subject. [0011] In some embodiments of the methods disclosed herein, the need for RBC transfusions and infusion platelet transfusion, intravenous immunoglobin (IVIG) transfusion, plasma transfusion and/or granulocyte transfusion in the subject is reduced or eliminated.

[0012] In some embodiments of the methods disclosed herein, the number and/or percent of F cells in the subject is modified following administration of the composition comprising the genetically modified cells.

[0013] In some embodiments of the methods disclosed herein, the genetically modified cell(s) are autologous or allogeneic. In some embodiments, the BCL11 A-genetically modified cells further comprise one or more additional genetic modifications.

In some embodiments of the methods disclosed herein, the genetically modified cells are hematopoietic stem cells isolated from the subject. In some embodiments, the hematopoietic stem cells are CD34+ hematopoietic stem or precursor cells (HSC/PC). In some embodiments, the CD34+ HSC/PC are mobilized in the subject by treatment with one or more doses of plerixafor. In some embodiments, at least 25 x 10⁶ CD34+ HSPCs/kg are mobilized in the subject. In some embodiments, the mobilized cells are harvested by one or more apheresis cycles.

[0014] In some embodiments of the methods disclosed herein, the methods further comprise administering one or more myeloablative condition agents to the subject prior to administration of the composition comprising the genetically modified cells. In some embodiments, the myeloablative agent comprises busulfan.

[0015] In some embodiments of the methods disclosed herein, the dose of genetically modified cells administered to the subject is between 3 x 10⁶ cells/kg and 20 x 10⁶ cells/kg. In some embodiments of the methods disclosed herein, the dose of cells administered to the subject is between 3 x 10⁶ cells/kg and 20 x 10⁶ cells/kg. In some embodiments of the methods disclosed herein, the dose of genetically modified cells administered to the subject is about 3.2 x 10⁶ cells/kg and about 9.7 x 10⁶ cells/kg. In some embodiments of the methods disclosed herein, the dose of genetically modified cells administered to the subject is about 5.17 x 10⁶ cells/kg.

[0016] In some embodiments of the methods disclosed herein, the genetically modified cells administered to the subject are formulated with approximately 1.0-2.0 x 10⁸ cells per bag at a concentration of approximately 1 x 10⁷ cells/mL.

[0017] In some embodiments of the methods disclosed herein, the genetically modified cells are cryopreserved prior to administration to the subject.

[0018] In some embodiments, the methods disclosed herein further comprise monitoring the subject’s vital signs prior to, during and/or after administration of the genetically modified cells. In some embodiments, the methods further comprise assessing hemoglobin, neutrophil and/or platelet levels in the subject prior to administration of the genetically modified cells to determine baseline levels of hemoglobin in the subject.

[0019] In some embodiments of the methods disclosed herein, the hemoglobin, neutrophil and/or platelet levels in the subject after administration of the genetically modified cells increase as compared to baseline levels for weeks or months after administration. In some embodiments, the hemoglobin, neutrophil and/or platelet levels in the subject remain stable as compared to baseline levels for weeks or months after administration.

[0020] In some embodiments, the methods disclosed herein further comprise assessing the subject using a health-related quality of life (HRQoL) survey. In some embodiments, the methods disclosed herein further comprise assessing the subject using the Patient-Reported Outcomes Measurement Information System 57 (PROMIS-57). In some embodiments, the methods disclosed herein further comprise assessing the subject using the Karnofsky Performance Scale.

[0021] In some embodiments of the methods disclosed herein, the subject receives one or more packed red blood cell (PRBC) transfusions prior to and/or after administration of the genetically modified cells.

[0022] In some embodiments of the methods disclosed herein, the need for additional therapies such as a bone marrow transplant, blood component and/or iron chelation therapy PRBC transfusions in the subject is reduced or eliminated. In some embodiments, the need for additional therapies is reduced or eliminated within 1-20 days of administration of the genetically modified cells. In some embodiments, the need for additional therapies in the subject is reduced or eliminated within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more days after administration of the genetically modified cells.

[0023] In another aspect, disclosed herein is a pharmaceutical composition comprising a genetically modified cell comprising a red blood cell (RBC) precursor cell, wherein the precursor cell comprises SB-mRENH1 mRNAs and SB-mRENH2 mRNAs, which encode a ZFN pair; and a genomic modification made following cleavage by the ZFN pair, wherein the modification is within an endogenous BCL11A enhancer sequence, and wherein the composition is formulated in cryopreservation buffer. In some embodiments, the cryopreservation buffer is CryoStor® CS-10 cryomedia.

[0024] These and other aspects will be readily apparent to the skilled artisan in light of disclosure as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 is an illustration (adapted from Hardison & Blobel (2013) Science 342(6155):206-7) of effects of low, elevated and high fetal hemoglobin levels on subjects comprising adult hemoglobin mutations (for example sickle cell disease). Shown on the far left (“low fetal hemoglobin:”) is a subject with a mutation in adult hemoglobin and wild-type ESE BCL11A, in this case the subject has normal (low) levels of fetal hemoglobin, resulting in disease symptoms in the subject. In the middle (“elevated fetal hemoglobin”), the subject has the adult hemoglobin mutation, but also has mutations in their BCL11A gene such that BCL11A expression is decreased but not eliminated, which results in elevated fetal globin levels. The subject experiences some disease amelioration due to the fetal globin “replacing” some adult globin functioning. In the far right (“high fetal hemoglobin), the subject has the adult globin mutation but has a deletion in the BCL11 A enhancer, such that the subject exhibits full expression of fetal globin. This subject will experience even greater in symptom improvement by virtue of full BCL11A inactivation.

[0026] FIG. 2 is a schematic depicting a treatment protocol using genetically modified HSPC (also referred to as SAR445136). “HSPC” refers to hematopoietic stem progenitor cells; “IV” refers to intravenous; “RBC” refers to red blood cells; and “ZFN” refers to zinc finger nuclease.

[0027] FIG. 3 is a schematic depicting the design of the clinical study. For a detailed schedule of events (SOE), see Tables 3 and 4.

[0028] FIG. 4 is a graph showing total Hb and Hb fractionation in four subjects after SAR445136 infusion. “HbA” refers to adult hemoglobin; “HbA2” refers to variant adult hemoglobin; “HbF” refers to fetal hemoglobin; and “HbS” refers to sickle hemoglobin.

[0029] FIG. 5 is a graph showing percentage of F cells over time for treated subjects. Further data remaining to be collected is noted by an asterisk (*).

[0030] FIG. 6 is a graph depicting HbF/F cell levels above the threshold range for preventing HbS polymerization for all four treated subjects. HbF: fetal hemoglobin. Further data remaining to be collected is noted by an asterisk (*).

[0031] FIG. 7 is a graphical depiction of the number of severe vaso-occlusive crises (VOCs) reported pre- and post-SAR445136 infusion in all four subjects.

DETAILED DESCRIPTION

[0032] Disclosed herein are methods for treating and/or preventing sickle cell disease events in a subject in need thereof. The present disclosure provides methods and compositions for genome editing and/or gene transfer. The present disclosure also provides methods and compositions for cell therapy for the treatment of subjects lacking sufficient expression of beta globin. In some embodiments, the methods and compositions disclosed herein are used to treat a subject with SCD comprising administering cells that have been modified using engineered nucleases to the subject. [0033] Also disclosed herein is SAR445136, which is a novel therapeutic product consisting of autologous CD34+ HSPCs that have been modified ex vivo by zinc finger nucleases (ZFN), targeting the BCL11A gene erythroid-specific enhancer (ESE) to increase endogenous production of HbF.

Definitions

[0034] As used herein, the term “approximately” or “about” as applied to one or more values of interest refers to a value that is similar to a stated reference value. In some embodiments, the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context.

[0035] The phrase “and/or” as used in the present disclosure will be understood to mean any one of the recited members individually or a combination of any two or more thereof - for example, “A, B, and/or C” would mean “A, B, C, A and B, A and C, B and C, or the combination of A, B, and C.”

[0036] The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” are used interchangeably and refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form. For the purposes of the present disclosure, these terms are not to be construed as limiting with respect to the length of a polymer. The terms can encompass known analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties (e.g., phosphorothioate backbones). In general, an analogue of a particular nucleotide has the same base-pairing specificity; /.e., an analogue of A will base-pair with T.

[0037] The terms “polypeptide,” “peptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of corresponding naturally-occurring amino acids.

[0038] “Binding” refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific. Such interactions are generally characterized by a dissociation constant (Kd) of 10^-6 M^-1 or lower. “Affinity” refers to the strength of binding: increased binding affinity being correlated with a lower Kd.

[0039] A “binding protein” is a protein that is able to bind non-covalently to another molecule. A binding protein can bind to, for example, a DNA molecule (a DNA-binding protein), an RNA molecule (an RNA-binding protein) and/or a protein molecule (a protein- binding protein). In the case of a protein-binding protein, it can bind to itself (to form homodimers, homotrimers, etc.) and/or it can bind to one or more molecules of a different protein or proteins. A binding protein can have more than one type of binding activity. For example, zinc finger proteins have DNA-binding, RNA-binding and protein-binding activity.

[0040] A “zinc finger DNA binding protein” (or binding domain) is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP. The term “zinc finger nuclease” includes one ZFN as well as a pair of ZFNs (the members of the pair are referred to as “left and right” or “first and second” or “pair”) that dimerize to cleave the target gene.

[0041] A “TALE DNA binding domain” or “TALE” is a polypeptide comprising one or more TALE repeat domains/units. The repeat domains are involved in binding of the TALE to its cognate target DNA sequence. A single “repeat unit” (also referred to as a “repeat”) is typically 33-35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. See, e.g., U.S. Patent Nos. 8,586,526 and 9,458,205. The term “TALEN” includes one TALEN as well as a pair of TALENs (the members of the pair are referred to as “left and right” or “first and second” or “pair”) that dimerize to cleave the target gene. Zinc finger and TALE binding domains can be “engineered” to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the recognition helix region of a naturally occurring zinc finger or TALE protein. Therefore, engineered DNA binding proteins (zinc fingers or TALEs) are proteins that are non-naturally occurring. Non-limiting examples of methods for engineering DNA-binding proteins are design and selection. A designed DNA binding protein is a protein not occurring in nature whose design/composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP and/or TALE designs and binding data. See, for example, U.S. Patent Nos. 8,568,526; 6,140,081 ; 6,453,242; and 6,534,261 ; see also International Patent Publication Nos. WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536; and WO 03/016496.

[0042] A “selected” zinc finger protein or TALE is a protein not found in nature whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection. See e.g., U.S. Patent Nos. 8,586,526; 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,200,759; and International Patent Publication Nos. WO 95/19431 ; WO 96/06166; WO 98/53057; WO 98/54311 ; WO 00/27878; WO 01/60970; WO 01/88197 and WO 02/099084. [0043] “Recombination” refers to a process of exchange of genetic information between two polynucleotides. For the purposes of this disclosure, “homologous recombination (HR)” refers to the specialized form of such exchange that takes place, for example, during repair of double-strand breaks in cells via homology-directed repair mechanisms. This process requires nucleotide sequence homology, uses a “donor” molecule to template repair of a “target” molecule (/.e., the one that experienced the double-strand break), and is variously known as “non-crossover gene conversion” or “short tract gene conversion,” because it leads to the transfer of genetic information from the donor to the target. Without wishing to be bound by any particular theory, such transfer can involve mismatch correction of heteroduplex DNA that forms between the broken target and the donor, and/or “synthesis-dependent strand annealing,” in which the donor is used to re-synthesize genetic information that will become part of the target, and/or related processes. Such specialized HR often results in an alteration of the sequence of the target molecule such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.

[0044] In the methods of the disclosure, one or more targeted nucleases as described herein create a double-stranded break in the target sequence {e.g., cellular chromatin) at a predetermined site, and a “donor” polynucleotide, having homology to the nucleotide sequence in the region of the break, can be introduced into the cell. The presence of the double-stranded break has been shown to facilitate integration of the donor sequence. The donor sequence may be physically integrated or, alternatively, the donor polynucleotide is used as a template for repair of the break via homologous recombination, resulting in the introduction of all or part of the nucleotide sequence as in the donor into the cellular chromatin. Thus, a first sequence in cellular chromatin can be altered and, in some embodiments, can be converted into a sequence present in a donor polynucleotide. Thus, the use of the terms “replace” or “replacement” can be understood to represent replacement of one nucleotide sequence by another, (/.e., replacement of a sequence in the informational sense), and does not necessarily require physical or chemical replacement of one polynucleotide by another.

[0045] In any of the methods described herein, additional pairs of zinc-finger or TALEN proteins can be used for additional double-stranded cleavage of additional target sites within the cell.

[0046] In some embodiments of methods for targeted recombination and/or replacement and/or alteration of a sequence in a region of interest in cellular chromatin, a chromosomal sequence is altered by homologous recombination with an exogenous “donor” nucleotide sequence. Such homologous recombination is stimulated by the presence of a doublestranded break in cellular chromatin, if sequences homologous to the region of the break are present. [0047] In any of the methods described herein, the first nucleotide sequence (the “donor sequence”) can contain sequences that are homologous, but not identical, to genomic sequences in the region of interest, thereby stimulating homologous recombination to insert a non-identical sequence in the region of interest. Thus, in some embodiments, portions of the donor sequence that are homologous to sequences in the region of interest exhibit between about 80 to 99% (or any integer therebetween) sequence identity to the genomic sequence that is replaced. In other embodiments, the homology between the donor and genomic sequence is higher than 99%, for example if only 1 nucleotide differs as between donor and genomic sequences of over 100 contiguous base pairs. In certain cases, a non-homologous portion of the donor sequence can contain sequences not present in the region of interest, such that new sequences are introduced into the region of interest. In these instances, the non-homologous sequence is generally flanked by sequences of 50-1 ,000 base pairs (or any integral value therebetween) or any number of base pairs greater than 1 ,000, that are homologous or identical to sequences in the region of interest. In other embodiments, the donor sequence is non-homologous to the first sequence and is inserted into the genome by non-homologous recombination mechanisms.

[0048] Any of the methods described herein can be used for partial or complete inactivation of one or more target sequences in a cell by targeted integration of donor sequence that disrupts expression of the gene(s) of interest. Cell lines with partially or completely inactivated genes are also provided.

[0049] Furthermore, the methods of targeted integration as described herein can also be used to integrate one or more exogenous sequences. The exogenous nucleic acid sequence can comprise, for example, one or more genes or cDNA molecules, or any type of coding or non-coding sequence, as well as one or more control elements (e.g., promoters). In addition, the exogenous nucleic acid sequence may produce one or more RNA molecules (e.g., small hairpin RNAs (shRNAs), inhibitory RNAs (RNAis), microRNAs (miRNAs), etc.).

[0050] “Cleavage” refers to the breakage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and doublestranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends. In some embodiments, fusion polypeptides are used for targeted double-stranded DNA cleavage.

[0051] A “cleavage half-domain” is a polypeptide sequence which, in conjunction with a second polypeptide (either identical or different) forms a complex having cleavage activity (preferably double-strand cleavage activity). The terms “first and second cleavage half- domains;” “+ and - cleavage half-domains” and “right and left cleavage half-domains” are used interchangeably to refer to pairs of cleavage half-domains that dimerize.

[0052] An “engineered cleavage half-domain” is a cleavage half-domain that has been modified so as to form obligate heterodimers with another cleavage half-domain (e.g., another engineered cleavage half-domain). See, U.S. Patent Nos. 7,888,121 ; 7,914,796; 8,034,598; and 8,823,618, incorporated herein by reference in their entireties.

[0053] The term “sequence” refers to a nucleotide sequence of any length, which can be DNA or RNA; can be linear, circular or branched and can be either single-stranded or double stranded. The term “donor sequence” refers to a nucleotide sequence that is inserted into a genome. A donor sequence can be of any length, for example between 2 and 10,000 nucleotides in length (or any integer value therebetween or thereabove), preferably between about 100 and 1 ,000 nucleotides in length (or any integer therebetween), more preferably between about 200 and 500 nucleotides in length.

[0054] A “disease associated gene” is one that is defective in some manner in a monogenic disease. Non-limiting examples of monogenic diseases include severe combined immunodeficiency, cystic fibrosis, lysosomal storage diseases (e.g., Gaucher’s, Hurler’s Hunter’s, Fabry’s, Neimann-Pick, Tay-Sach’s, etc.), sickle cell anemia, and thalassemia.

[0055] A “chromosome” is a chromatin complex comprising all or a portion of the genome of a cell. The genome of a cell is often characterized by its karyotype, which is the collection of all the chromosomes that comprise the genome of the cell. The genome of a cell can comprise one or more chromosomes.

[0056] An “episome” is a replicating nucleic acid, nucleoprotein complex or other structure comprising a nucleic acid that is not part of the chromosomal karyotype of a cell. Examples of episomes include plasmids and certain viral genomes.

[0057] A “target site” or “target sequence” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided sufficient conditions for binding exist.

[0058] An “exogenous” molecule is a molecule that is not normally present in a cell, but can be introduced into a cell by one or more genetic, biochemical or other methods. “Normal presence in the cell” is determined with respect to the particular developmental stage and environmental conditions of the cell. Thus, for example, a molecule that is present only during embryonic development of muscle is an exogenous molecule with respect to an adult muscle cell. Similarly, a molecule induced by heat shock is an exogenous molecule with respect to a non-heat-shocked cell. An exogenous molecule can comprise, for example, a functioning version of a malfunctioning endogenous molecule or a malfunctioning version of a normally- functioning endogenous molecule. [0059] An exogenous molecule can be, among other things, a small molecule, such as is generated by a combinatorial chemistry process, or a macromolecule such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules. Nucleic acids include DNA and RNA, can be single- or double-stranded; can be linear, branched or circular; and can be of any length. Nucleic acids include those capable of forming duplexes, as well as triplex-forming nucleic acids. See, for example, U.S. Patent Nos. 5,176,996 and 5,422,251. Proteins include, but are not limited to, DNA-binding proteins, transcription factors, chromatin remodeling factors, methylated DNA binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases and helicases.

[0060] An exogenous molecule can be the same type of molecule as an endogenous molecule, e.g., an exogenous protein or nucleic acid. For example, an exogenous nucleic acid can comprise an infecting viral genome, a plasmid or episome introduced into a cell, or a chromosome that is not normally present in the cell. Methods for the introduction of exogenous molecules into cells are known to those of skill in the art and include, but are not limited to, lipid-mediated transfer (/.e., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE- dextran-mediated transfer and viral vector-mediated transfer. An exogenous molecule can also be the same type of molecule as an endogenous molecule but derived from a different species than the cell is derived from. For example, a human nucleic acid sequence may be introduced into a cell line originally derived from a mouse or hamster.

[0061] By contrast, an “endogenous” molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions. For example, an endogenous nucleic acid can comprise a chromosome, the genome of a mitochondrion, chloroplast or other organelle, or a naturally-occurring episomal nucleic acid. Additional endogenous molecules can include proteins, for example, transcription factors and enzymes.

[0062] A “fusion” molecule is a molecule in which two or more subunit molecules are linked, preferably covalently. The subunit molecules can be the same chemical type of molecule, or can be different chemical types of molecules. Examples of fusion molecules include, but are not limited to, fusion proteins (for example, a fusion between a protein DNA- binding domain and a cleavage domain), fusions between a polynucleotide DNA-binding domain (e.g., sgRNA) operatively associated with a cleavage domain, and fusion nucleic acids (for example, a nucleic acid encoding the fusion protein). [0063] Expression of a fusion protein in a cell can result from delivery of the fusion protein to the cell or by delivery of a polynucleotide encoding the fusion protein to a cell, wherein the polynucleotide is transcribed, and the transcript is translated, to generate the fusion protein. Trans-splicing, polypeptide cleavage and polypeptide ligation can also be involved in expression of a protein in a cell. Methods for polynucleotide and polypeptide delivery to cells are presented elsewhere in this disclosure.

[0064] A “gene,” for the purposes of the present disclosure, includes a DNA region encoding a gene product (see infra), as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions.

[0065] “Gene expression” refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.

[0066] “Modulation” of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression. Genome editing {e.g., cleavage, alteration, inactivation, random mutation) can be used to modulate expression. Gene inactivation refers to any reduction in gene expression as compared to a cell that does not include a ZFP or TALEN as described herein. Thus, gene inactivation may be partial or complete.

[0067] A “region of interest” is any region of cellular chromatin, such as, for example, a gene or a non-coding sequence within or adjacent to a gene, in which it is desirable to bind an exogenous molecule. Binding can be for the purposes of targeted DNA cleavage and/or targeted recombination. A region of interest can be present in a chromosome, an episome, an organellar genome (e.g., mitochondrial, chloroplast), or an infecting viral genome, for example. A region of interest can be within the coding region of a gene, within transcribed non-coding regions such as, for example, leader sequences, trailer sequences or introns, or within non-transcribed regions, either upstream or downstream of the coding region. A region of interest can be as small as a single nucleotide pair or up to 2,000 nucleotide pairs in length, or any integral value of nucleotide pairs. [0068] “Eukaryotic” cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells (e.g., stem cells, or precursor cells). The term “stem cells” or “precursor cells” refer to pluripotent and multipotent stem cells, including but not limited to hematopoietic stem cells, which are also referred to as hematopoietic progenitor stem cells (HPSC) or hematopoietic stem cell/precursor cells (HSC/PC).

[0069] “Red Blood Cells” (RBCs) or erythrocytes are terminally differentiated cells derived from hematopoietic stem cells. They lack a nuclease and most cellular organelles. RBCs contain hemoglobin to carry oxygen from the lungs to the peripheral tissues. In fact, 33% of an individual RBC is hemoglobin. They also carry CO2 produced by cells during metabolism out of the tissues and back to the lungs for release during exhale. RBCs are produced in the bone marrow in response to blood hypoxia which is mediated by release of erythropoietin (EPO) by the kidney. EPO causes an increase in the number of proerythroblasts and shortens the time required for full RBC maturation. After approximately 120 days, since the RBC do not contain a nucleus or any other regenerative capabilities, the cells are removed from circulation by either the phagocytic activities of macrophages in the liver, spleen and lymph nodes (-90%) or by hemolysis in the plasma (-10%). Following macrophage engulfment, chemical components of the RBC are broken down within vacuoles of the macrophages due to the action of lysosomal enzymes.

[0070] The terms “operative linkage” and “operatively linked” (or “operably linked”) are used interchangeably with reference to a juxtaposition of two or more components (such as sequence elements), in which the components are arranged such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. By way of illustration, a transcriptional regulatory sequence, such as a promoter, is operatively linked to a coding sequence if the transcriptional regulatory sequence controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. A transcriptional regulatory sequence is generally operatively linked in cis with a coding sequence, but need not be directly adjacent to it. For example, an enhancer is a transcriptional regulatory sequence that is operatively linked to a coding sequence, even though they are not contiguous.

[0071] With respect to fusion polypeptides, the term “operatively linked” can refer to the fact that each of the components performs the same function in linkage to the other component as it would if it were not so linked. For example, with respect to a fusion polypeptide in which a ZFP or TALE DNA-binding domain is fused to an activation domain, the ZFP or TALE DNA- binding domain and the activation domain are in operative linkage if, in the fusion polypeptide, the ZFP or TALE DNA-binding domain portion is able to bind its target site and/or its binding site, while the activation domain is able to up-regulate gene expression. When a fusion polypeptide in which a ZFP or TALE DNA-binding domain is fused to a cleavage domain, the ZFP or TALE DNA-binding domain and the cleavage domain are in operative linkage if, in the fusion polypeptide, the ZFP or TALE DNA-binding domain portion is able to bind its target site and/or its binding site, while the cleavage domain is able to cleave DNA in the vicinity of the target site.

[0072] A “functional” protein, polypeptide or nucleic acid includes any protein, polypeptide or nucleic acid that provides the same function as the wild-type protein, polypeptide or nucleic acid. A “functional fragment” of a protein, polypeptide or nucleic acid is a protein, polypeptide or nucleic acid whose sequence is not identical to the full-length protein, polypeptide or nucleic acid, yet retains the same function as the full-length protein, polypeptide or nucleic acid. A functional fragment can possess more, fewer, or the same number of residues as the corresponding native molecule, and/or can contain one or more amino acid or nucleotide substitutions. Methods for determining the function of a nucleic acid (e.g., coding function, ability to hybridize to another nucleic acid) are well-known in the art. Similarly, methods for determining protein function are well-known. For example, the DNA-binding function of a polypeptide can be determined, for example, by filter-binding, electrophoretic mobility-shift, or immunoprecipitation assays. DNA cleavage can be assayed by gel electrophoresis. See, Ausubel et al., supra. The ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, two-hybrid assays or complementation, both genetic and biochemical. See, for example, Fields et al. (1989) Nature 340:245-246; U.S. Patent No. 5,585,245 and International Patent Publication No. WO 98/44350.

[0073] A “vector” is capable of transferring gene sequences to target cells. Typically, “vector construct,” “expression vector,” and “gene transfer vector,” mean any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells. Thus, the term includes cloning, and expression vehicles, as well as integrating vectors.

[0074] A “reporter gene” or “reporter sequence” refers to any sequence that produces a protein product that is easily measured, preferably although not necessarily in a routine assay. Suitable reporter genes include, but are not limited to, sequences encoding proteins that mediate antibiotic resistance (e.g., ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance), sequences encoding colored or fluorescent or luminescent proteins (e.g., green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, luciferase), and proteins which mediate enhanced cell growth and/or gene amplification (e.g., dihydrofolate reductase). Epitope tags include, for example, one or more copies of FLAG, His, myc, Tap, HA or any detectable amino acid sequence. “Expression tags” include sequences that encode reporters that may be operably linked to a desired gene sequence in order to monitor expression of the gene of interest.

[0075] The terms “subject” and “patient” are used interchangeably and refer to mammals such as human subjects and non-human primates, as well as experimental animals such as rabbits, dogs, cats, rats, mice, and other animals. Accordingly, the term “subject” or “patient” as used herein means any mammalian subject or patient to which the altered cells of the invention and/or proteins produced by the altered cells of the invention can be administered. In some embodiments, the subject has a confirmed diagnosis of SCD. In some embodiments, the subject has a confirmed diagnosis of severe SCD.

[0076] As used herein, “SCD” or “sickle cell disease” refers to a disease characterized by the presence of hemoglobin S (Hb S), either from homozygosity for the sickle mutation (Hb SS) or compound heterozygosity with another beta globin variant (eg, sickle-beta thalassemia, Hb SC disease). The hallmarks of SCD include vaso-occlusive phenomena and hemolytic anemia.

[0077] As used herein “severe SCD” or “severe sickle cell disease” refers to a sickle cell disease having one or more of the following manifestations consistent with Bone Marrow Transplant Clinical Trial Network (BMT CTN) guidelines: (1) clinically significant neurologic event (stroke) or any neurological deficit lasting >24 hours; (2) history of 2 or more episodes of ACS in the 2-year period preceding informed consent despite adequate supportive care measures (i.e. , asthma therapy); (3) three or more pain crises per year in the 2-year period preceding informed consent (requiring IV pain management in the outpatient or inpatient hospital setting); (4) history of two or more episodes of priapism with subject seeking medical care in the 2-year period preceding informed consent; (5) administration of regular RBC transfusion therapy in the year preceding informed consent, defined as receiving 8 or more transfusions to prevent vaso-occlusive clinical complications (i.e., pain, stroke, or ACS); and/or (6) an echocardiographic finding of tricuspid valve regurgitant jet (TRJ) velocity >2.5 m/s.

[0078] In some embodiments, the subjects disclosed herein are eligible for treatment for sickle cell disease. For the purposes herein, such a subject can be one who is experiencing, has experienced, or is likely to experience, one or more signs, symptoms or other indicators of sickle cell disease; has been diagnosed with sickle cell disease, whether, for example, newly diagnosed, and/or is at risk for developing sickle cell disease. One suffering from or at risk for suffering from sickle cell disease may optionally be identified as one who has been screened for abnormally low levels of hemoglobin in their blood or plasma.

[0079] As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: decreasing one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), delay or slowing the progression of the disease, ameliorating the disease state, decreasing the dose of one or more other medications required to treat the disease, and/or increasing the quality of life.

[0080] As used herein, “delaying” or “slowing” the progression of sickle cell disease means to prevent, defer, hinder, slow, retard, stabilize, and/or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated.

[0081] As used herein, “at the time of starting treatment” refers to the time period at or prior to the first exposure to a sickle cell disease therapeutic composition such as the compositions disclosed herein. In some embodiments, “at the time of starting treatment” is about any of one year, nine months, six months, three months, second months, or one month prior to a sickle cell disease drug. In some embodiments, “at the time of starting treatment” is immediately prior to coincidental with the first exposure to a sickle cell disease therapeutic composition.

[0082] As used herein, “based upon” includes (1) assessing, determining, or measuring the subject characteristics as described herein (and preferably selecting a subject suitable for receiving treatment; and (2) administering the treatment(s) as described herein.

[0083] A “symptom” of sickle cell disease is any phenomenon or departure from the normal in structure, function, or sensation, experienced by the subject and indicative of sickle cell disease.

[0084] The term “supportive surgery” refers to surgical procedures that may be performed on a subject to alleviate symptoms that may be associated with a disease.

[0085] The term “immunosuppressive agent” as used herein for adjunct therapy refers to substances that act to suppress or mask the immune system of the mammal being treated herein. This would include substances that suppress cytokine production, down-regulate or suppress self-antigen expression, or mask the MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines (see, U.S. Patent No. 4,665,077); nonsteroidal anti-inflammatory drugs (NSAIDUA); ganciclovir, tacrolimus, glucocorticoids such as cortisol or aldosterone, anti-inflammatory agents such as a cyclooxygenase inhibitor, a 5 - lipoxygenase inhibitor, or a leukotriene receptor antagonist; purine antagonists such as azathioprine or mycophenolate mofetil (MMF); alkylating agents such as cyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde (which masks the MHC antigens, as described in U.S. Patent No. 4,120,649); anti-idiotypic antibodies for MHC antigens and MHC fragments; cyclosporin A; steroids such as corticosteroids or glucocorticosteroids or glucocorticoid analogs, e.g., prednisone, methylprednisolone, and dexamethasone; dihydrofolate reductase inhibitors such as methotrexate (oral or subcutaneous); hydroxychloroquine; sulfasalazine; leflunomide; cytokine or cytokine receptor antagonists including anti-interferon-alpha, -beta, or -gamma antibodies, anti-tumor necrosis factor-alpha antibodies (infliximab or adalimumab), anti-TNF-alpha immunoahesin (etanercept), anti-tumor necrosis factor-beta antibodies, anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies; anti-LFA-1 antibodies, including anti-CD11a and anti-CD18 antibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-T antibodies, preferably anti-CD3 or anti- CD4/CD4a antibodies; soluble peptide containing a LFA-3 binding domain (International Patent Publication No. WO 90/08187 published 7/26/90); streptokinase; TGF-beta; streptodornase; RNA or DNA from the host; FK506; RS-61443; deoxyspergualin; rapamycin; T-cell receptor (Cohen et al., U.S. Patent No. 5,114,721); T-cell receptor fragments (Offner et al. (1991) Science 251 :430-432; International Patent Publication No. WO 90/11294; Janeway (1989) Nature 341 :482; and International Patent Publication No. WO 91/01133); and T cell receptor antibodies such as T10B9.

[0086] “Corticosteroid” refers to any one of several synthetic or naturally occurring substances with the general chemical structure of steroids that mimic or augment the effects of the naturally occurring corticosteroids. Examples of synthetic corticosteroids include prednisone, prednisolone (including methylprednisolone), dexamethasone, glucocorticoid and betamethasone.

[0087] “Iron chelation” is a type of therapy to remove excess iron from the body. Each unit of blood given in a transfusion comprises about 250 milligrams of iron, and the body cannot excrete it except in small (~1 mg) amounts that are lost in skin and perspiration. Excess iron is trapped in the tissues of vital organs, such as the anterior pituitary, heart, liver, pancreas and joints. When the iron reaches toxic levels, damage can result in diseases such as diabetes, cirrhosis, osteoarthritis, heart attack, and hormone imbalances. Hypothyroidism, hypogonadism, infertility, impotence and sterility can result from these hormone imbalances. If not addressed, excess iron can result in complete organ failure and death. Iron reduction is accomplished with chelation therapy, which is the removal of iron pharmacologically with an iron-chelating agent such as desferrioxamine or deferasirox.

[0088] A “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications, other therapeutic products to be combined with the packaged product, and/or warnings concerning the use of such therapeutic products, etc. [0089] A “label” is used herein to refer to information customarily included with commercial packages of pharmaceutical formulations including containers such as vials and package inserts, as well as other types of packaging.

[0090] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of skill in the art.

[0091] Practice of the methods, as well as preparation and use of the compositions disclosed herein employ, unless otherwise indicated, conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, computational chemistry, cell culture, recombinant DNA and related fields as are within the skill of the art. These techniques are fully explained in the literature. See, for example, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001 ; Ausubel et a/., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, “Chromatin” (P.M. Wassarman and A. P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, “Chromatin Protocols” (P.B. Becker, ed.) Humana Press, Totowa, 1999.

Methods of treatment

[0092] In some embodiments, the subject has received a diagnosis of severe SCD. Severe SCD was defined as having 1 or more of the following manifestations consistent with Bone Marrow Transplant Clinical Trial Network (BMT CTN) guidelines: (1) clinically significant neurologic event (stroke) or any neurological deficit lasting >24 hours; (2) history of 2 or more episodes of ACS in the 2-year period preceding informed consent despite adequate supportive care measures (i.e. , asthma therapy); (3) three or more pain crises per year in the 2-year period preceding informed consent (requiring IV pain management in the outpatient or inpatient hospital setting); (4) history of two or more episodes of priapism with subject seeking medical care in the 2-year period preceding informed consent; (5) administration of regular RBC transfusion therapy in the year preceding informed consent, defined as receiving 8 or more transfusions to prevent vaso-occlusive clinical complications (i.e., pain, stroke, or ACS); and/or (6) an echocardiographic finding of tricuspid valve regurgitant jet (TRJ) velocity >2.5 m/s. [0093] In some embodiments, the subject is required to have adequate physiological function consistent with BMT CNT guidelines to receive the treatments described herein. As a non-limiting example, in certain embodiments, adequate physiological function may be measured by (a) Karnofsky/Lansky Performance Scale score >60; (b) cardiac function - left ventricular ejection fraction (LVEF) >40% or left ventricular shortening fraction >26% by cardiac echocardiogram (ECHO) or by multiple gated acquisition (MLIGA) scan (c) pulmonary function - pulse oximetry with a baseline oxygen (02) saturation of >85% and diffusing capacity for carbon monoxide (DLCO) >50% (corrected for hemoglobin); and (d) hepatic function - alanine aminotransferase (ALT) and aspartate aminotransferase (AST) <5* the upper limit of normal as per local laboratory: serum conjugated (direct) bilirubin <3* the upper limit of normal for age as per local laboratory. If direct bilirubin is >0.5 mg/dL, subject must have either a liver magnetic resonance imaging (MRI) with a liver iron content (LIC) <10 mg/g dry weight, or a liver biopsy demonstrating absence of bridging fibrosis, liver cirrhosis, and active hepatitis in order to participate (results of liver biopsies obtained within the past 12 months are allowed).

[0094] In some embodiments, described herein are modified autologous HSC/PC that are delivered to a subject with SCD according to the disclosed methods. Two mRNAs encoding the right and left ZFN partners are delivered to the harvested HSC/PC which are targeted to the BCL11a erythroid enhancer sequence. In some embodiments, the mRNAs include SB- mRENHI and SB-mRENH2. In some embodiments, the CD34+ HSC/PCs are harvested {e.g., apheresis) after mobilization in the subject by treating the subject with one or more doses of plerixafor prior to isolation and the mobilized cells. In some embodiments, at least about 25 x 10⁶ CD34+ HSPCs/kg are harvested in total or per apheresis cycle and may be cultured for any length of time. The resulting genetically modified cells may be cultured and descendants thereof will include the specific BCL11A genetic modification {e.g., less than 1% of cells having off-target (non-BCL11 A) modifications), but not necessarily the mRNA(s). Cells comprising the BCL11A knockout can then be infused into the subjects. In some embodiments, additional modifications, such for example inactivation of HLA genes may be made in the specific BCL11A genetically modified cells.

[0095] In some embodiments, the methods described herein disclose genetically modified cells that are hematopoietic stem cells e.g., CD34+ HSC/PC) isolated from the subject, optionally in which the CD34+ HSC/PCs are mobilized {e.g., at least 25 x 10⁶ CD34+ HSPCs/kg) in each subject by treatment with one or more doses of plerixafor prior to isolation and the mobilized cells are harvested by one or more apheresis cycles.

[0096] Methods of altering expression of hemoglobin, including for use in the treatment of SCD, as provided herein, include methods that result in a change from baseline of clinical laboratory hemoglobin fractions (adult hemoglobin, HbA and fetal hemoglobin, HbF) in terms of both changes in grams/dL plasma and percent HbF of total Hb in a subject. See, e.g. FIG. 4.

[0097] In some embodiments, the methods of treatment described herein result in a change from baseline in the number and percent of F cells. F cells are RBCs that contain measurable amounts of HbF. Evaluation of a change in F cells as a result of the treatment methods can be measured by methods known in the art (see e.g., Wood et al. (1975) Blood 46(5):671). In some embodiments, the number and/or percentage of F cells is increased in a subject treated as described herein, as compared to an untreated subject.

[0098] HbF normally only plays a minor role in normal adult physiology. In patients with SCD, clinical studies demonstrate that mortality and disease severity correlate with HbF levels. See, e.g., Sankaran (2011). Am Soc Hematol Educ Program Book. 2011 :459-465. HbF achieves this disease amelioration by reducing HbS content and disrupting HbS polymer formation within the red cell. The effects of low, elevated, and high HbF levels are depicted in FIG. 1. Elevation of HbF concentration up to 20% is a primary mechanism for the activity of hydroxyurea (HU). Further, patients with concurrent SCD and persistent high levels of HbF (20% to 30%) due to the condition of hereditary persistence of fetal hemoglobin (HPFH) are asymptomatic throughout life. (Xu et al. (2011) Science 334(6058): 993-6). These studies support a therapeutic approach of increasing HbF as a mechanism for treating SCD.

[0099] In some embodiments, use of the methods of treatment disclosed herein may result in a change of SCD-related disease biomarkers. In some embodiments, changes in the SCD- related disease biomarkers may include, but are not limited to, changes in iron metabolism and/or changes in levels of erythropoietin, haptoglobin and hepcidin levels. In some embodiments, the methods of treatment may result in a change in a patient’s symptoms associated with iron overload associated with baseline transfusion therapy. Changes in iron overload symptoms may include a decrease in endocrine dysfunction caused by iron deposition in endocrine organs. Endocrine dysfunction may be evaluated by measurement of factors (levels and/or activity) such as, but not limited to, thyroid hormones, IGF-1 , morning cortisol, adrenocorticotropic hormone (ACTH), HbA1C, and/or vitamin D. Determination of all the above factors, including HbA, HbF, erythropoietin, haptoglobin, hepcidin, thyroid hormones, IGF-1 , cortisol, ACTH, and vitamin D may be measured by standard clinical laboratory protocols.

[0100] In some embodiments, provided herein are methods of treatment that reduce, delay, and/or eliminate additional treatment procedures as compared with a subject that has not been treated with the methods and compositions as disclosed herein, for example wherein an effective amount of modified HSC/PC are administered to a subject in need thereof, wherein the subject has a reduced, delayed, and/or eliminated need for additional treatment procedures after treatment. In some embodiments, the additional treatment procedures can include, but are not limited to, a bone marrow transplant, PRBC and/or other blood component transfusions, and treatments related to iron chelation therapy.

[0101] In some embodiments, the uses and methods of treatment described herein will result in a decrease in the need for (use of) RBC transfusions and infusion of other blood products including, but not limited to, platelets, intravenous immunoglobin (IVIG) , plasma and granulocytes in a subject with sickle cell disease. Change in the use of RBC and other blood product infusions in a subject treated with the methods and compositions of the invention can be evaluated by keeping a log of use for the subject. The log can be used to calculate an annualized frequency and volume of packed red blood cells (PRBC) after infusion with the compositions disclosed herein, and compared to the subject’s past PRBC and other blood products usage prior to treatment.

[0102] In some embodiments, disclosed herein is a method of reducing, delaying or eliminating the SCD-related disease biomarkers following treatment with the methods and compositions in a subject with SCD as compared with the subject prior to treatment with the methods and compositions disclosed herein. In some embodiments, the subject is administered an effective amount of modified HSC/PC such that the subject has reduced, delayed or eliminated SCD-related disease biomarkers after treatment. In some embodiments, levels of HbF increase by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400% or more (or any value therebetween) following treatment by the methods disclosed herein. In some embodiments, the effects of the disclosed methods on the subject are realized within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more days after administration of the genetically modified cells.

[0103] In another aspect, disclosed herein is a method of reducing, delaying or eliminating the use of PRBC transfusions and infusion of other blood products including, but not limited to, platelets, intravenous immunoglobin (IVIG), plasma and granulocytes following treatment with the methods and compositions in a subject with SCD as compared with a subject that has not been treated with the methods and compositions of the invention. In some embodiments, the use of PRBC and/or other blood product is decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any value therebetween in a subject treated with the methods disclosed herein as compared to the subject prior to receiving treatment. In some embodiments, the use of PRBC and/or other blood product infusions is eliminated.

In another aspect, disclosed herein is a method of reducing, delaying or eliminating the symptoms associated with iron overload in a subject with sickle cell disease. In some embodiments, markers of endocrine dysfunction as a result of iron deposition in endocrine organs (for example, thyroid markers, IGF-1 , morning cortisol, HbA1C and Vitamin D) become normalized in a subject after treatment with the methods and compositions of the invention as compared to the marker levels prior to treatment. In some embodiments, iron overload in the liver and heart is decreased in a subject following treatment with the methods and compositions disclosed herein as compared with the subject prior to treatment. Iron overload can be evaluated by standard MRI procedures. In some embodiments, iron over load in the liver and/or heart detected by MRI is decreased by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any value therebetween in a subject treated with the methods disclosed herein as compared to the subject prior to receiving treatment.

[0104] In some embodiments, ex vivo therapies for SCD are described using the genetically modified cells as described herein. In some embodiments, the genetically modified cells are autologous cells obtained from the subject to be treated, which cells are then genetically modified as described herein and administered back to the same subject. Cells obtained from the subject may be mobilized using treatment with plerixafor. See, e.g., llchida et al. (2020) Haematologica. 105(10): e497-e501. In the methods described herein, any amount of cells may be mobilized, for example about 5 x 10⁵, about 10 x 10⁵, about 15 x 10⁵, about 20 x 10⁵, about 5 x 10⁶, about 10 x 10⁶, about 15 x 10⁶, about 20 x 10⁶, about 25 x 10⁶ CD34+ HSPCs/kg for genetic modification are mobilized in the subject.

[0105] Plerixafor reversibly inhibits binding of stromal cell-derived factor-1-alpha (SDF- 1a), expressed on bone marrow stromal cells, to the CXC chemokine receptor 4 (CXCR4), resulting in mobilization of CD34+ HSPCs from bone marrow into peripheral blood. Plerixafor is indicated in combination with G-CSF to mobilize hematopoietic stem cells to the peripheral blood for collection and subsequent autologous transplantation in patients with multiple myeloma or non-Hodgkin's lymphoma. However, the use of G-CSF in patients with SCD is contraindicated due to risk of vaso-occlusive crisis. See, e.g., Tisdale JF et al. (2017) Blood 130 (S1): 990.

[0106] In some embodiments, mobilized CD34+ HSPCs are collected from each subject one or more days {e.g., 3, 4, 5, 6, 7 or more days) after mobilization by apheresis, for example on 2 or more consecutive days until sufficient cells are collected. In some embodiments, at least about 1 x 10⁴ to 1 x 10⁷ e.g., 25 x 10⁶) CD34+ HSPCs/kg are collected. If needed, a second mobilization and apheresis cycle may be performed 1 , 2, 3, or more weeks after the first cycle. In some embodiments, a portion of collected cells are subject to genetic modification as described herein and the remainder maintained {e.g., cryopreserved) in the event a rescue treatment for the subject is indicated.

[0107] In some embodiments, the modified HSPC are evaluated prior to returning to the subject. In some embodiments, the modified cells are evaluated for the presence and type of nuclease-induced mutations in the BCL11A enhancer region. In some embodiments, the mutations can be insertions of nucleotides, deletions of nucleotides or both (“indels”). In some embodiments, the cells are evaluated for off-target cleavage by the nucleases. In some embodiments, the cells are evaluated for insertions and/or deletions within BCL11 A (on-target modifications) and/or other non-BCL11A region (off-target modifications). In some embodiments, the cells are evaluated for molecular translocations and/or karyotyping of the cellular chromosomes following nuclease cleavage. In some embodiments, the cells are evaluated for off-target transcriptional activity. In some embodiments, the cells are evaluated for endotoxin load. In some embodiments, the cells can be evaluated for one or more of the above characteristics.

[0108] The autologous cells are genetically modified as described herein and cryopreserved e.g., using a controlled rate freezer) according to standard techniques with each aliquot {e.g., infusion bag) having a total cell count of approximately 1.0 x 10⁸ to 2.0 x 10⁸ cells and can be stored in vapor phase liquid nitrogen (at < -150°C) at the manufacturing facility until they are ready to be shipped to the site where they will be administered to a subject.

[0109] In certain aspects, the ex vivo therapies comprise thawing the frozen genetically modified HSPC and infusing the cells into the subject, preferably within about 15 to about 45 minutes of thawing. The volume of frozen modified HSPC administered is determined by the subject’s weight. Vital signs (blood pressure, temperature, heart rate, respiratory rate and pulse oximetry) can be monitored prior to infusion and afterwards. In some embodiments, the subjects are monitored using blood tests as well as analysis of HbF levels (baseline levels of HbF fractions (A and F in g/dL) and percent HbF is determined based on the last assessment on or prior to the date of first administration of IV busulfan), endocrine function, and/or performing MRIs to assess iron load. In some embodiments, the ex vivo therapies result in neutrophil and platelet recovery to within normal levels in the subject in about two to four weeks of infusion. Subjects may also receive PRBC transfusions 0, 1 , or more times following HSPC infusion. In some embodiments, total hemoglobin levels in the subject remain stable or continue to rise by week 2, 3, 4, 5, 6, 7, or more after infusion with the modified HSPC.

[0110] In any of the methods described herein, the subject can receive conditioning therapy prior to ex vivo therapy with genetically modified cells, for example, via intravenous (IV) administration of one or more myeloablative condition agents prior to infusion with the modified CD34+ HSC/PC. In some embodiments, the myeloablative condition agent is busulfan. In some embodiments, the busulfan is used with other agents such as cyclophosphamide. [0111] Busulfan can be administered using known effective doses and regimens. According to standard procedures, for example, busulfan is dosed at between about 0.5 to 5 mg/kg (or any value therebetween). In some embodiments, subjects will receive a myeloablative regimen of busulfan (about 3.2 mg/kg/day; IV via central venous catheter) for up to 4 days (total dose of about 12.8 mg/kg prior to infusion), for example on Days -6 through -3 before infusion of the modified HSPC on Day 0. IV busulfan may be dosed once daily (total of 4 doses) or every 6 hours (total of 16 doses) according to study center practices or guidelines. After the first dose, the IV busulfan dose can be adjusted based on pharmacokinetic sampling and study center practices to target an area under the curve (AUG) of 4,000-5,000 mmol*min for daily dosing or an AUG of 1 ,000-1 ,250 mmol*min for every 6 hour dosing for a total regimen target AUG of 16,000-20,000 mmol*min. IV busulfan pharmacokinetic targeting may be modified for subsequent subjects. Optionally, therapeutic drug monitoring is conducted to determine clearance of busulfan after 4 days of dosing is complete.

[0112] Any dose of genetically modified cells can be used, for example, between 3 x 10⁶ cells/kg and 20 x 10⁶ cells/kg e.g., where the cells are formulated with approximately 1 .0- 2.0 x 10⁸ cells per bag at a concentration of approximately 1 x 10⁷ cells/mL). In some embodiments, a dose of about 3 x 10⁶ cells/kg to about 20 x 10⁶ cells/kg (or any value therebetween) of the genetically modified cells is administered {e.g., via intravenous infusion) to the subject. In some embodiments, the cells are formulated in infusible cryomedia containing 10% DMSO. In some embodiments, the cells are formulated with approximately 1.0- 2.0 x 10⁸ cells per bag at a concentration of approximately 1 x 10⁷ cells/mL. In any of the methods described herein, cell dosages may be determined as total cell dose or as a CD34+ cell dose, which can be calculated as follows: CD34+ dose = [total cell dose] x [CD34+ %].

[0113] In some embodiments, the dose of genetically modified cells administered to the subject is between about 3.2 x 10⁶ cells/kg and 9.7 x 10⁶ cells/kg. In some embodiments, the dose of genetically modified cells administered to the subject is about 5.17 x 10⁶ cells/kg. In some embodiments, the dose of cells administered to the subject is between about 3.2 x 10⁶ cells/kg and 9.7 x 10⁶ cells/kg. In some embodiments, the dose of cells administered to the subject is about 5.17 x 10⁶ cells/kg.

[0114] In some embodiments, the subject has delayed, reduced or eliminated need, for example, for additional therapeutic procedures after receiving a total dose of between about 3.0 x 10⁶ to about 20 x 10⁶ cells/kg.

[0115] In some embodiments, the subject has delayed, reduced or eliminated need, for example, for additional therapeutic procedures after receiving a total dose of between 3.2 x 10⁶ cells/kg and 9.7 x 10⁶ cells/kg. In some embodiments, the subject has delayed, reduced or eliminated need, for example, for additional therapeutic procedures after receiving a total dose of about 5.17 x 10⁶ cells/kg.

[0116] In some embodiments, the methods and compositions disclosed herein comprise dosing of a composition of the invention (for example, the modified HSC/PC), for example, via a peripheral vein catheter. In some embodiments, the composition is administered to the subject which is then followed by administration of normal saline (NS) or phosphate buffered saline (PBS).

[0117] Following infusion, the modified HSPC may be monitored in the patient to determine engraftment efficiency and/or modification heterogenicity. This can be done, for example, by determining the genetic modification (“indel”) profile. Cell samples may be purified from the peripheral blood, bone marrow aspirate or other tissue samples (preferably about 5 x 10⁴ to 1 x 10⁷ cells) and subject to genomic DNA isolation for assessment. Bone marrow aspirate or other tissue samples may be taken at various timepoints, including at between about 6-9 months. In some embodiments, the indel profile is monitored over time to determine the likelihood of any one particular cell type (indel profile) aberrantly overgrowing the population. In some embodiments, the cells and subject are monitored before and/or after administration, for example to determine the indel profile of cells isolated from peripheral blood samples, bone marrow aspirates, or other tissue sources in comparison with the indel profile of the infused cells to in order to monitor stability of the graft in the subject. In some embodiments, genomic DNA from cells isolated from a treated subject is isolated and the region comprising the BCL11A target sequence is amplified. In further embodiments, the percent modified cells within the cell population is determined and re-tested over time post dosing to evaluate stability of the modified cell population with the treated subject.

[0118] The methods may further comprise monitoring the subject’s vital signs prior to, during and/or after administration of the genetically modified cells; and/or assessing hemoglobin, neutrophil and/or platelet levels in the subject prior to administration of the genetically modified cells to determine baseline levels of hemoglobin in the subject. In some embodiments, hemoglobin, neutrophil and/or platelet levels in the subject after administration of the genetically modified cells increase or remain stable as compared to baseline levels for weeks or months after administration.

[0119] Optionally, the subject may receive one or more PRBC transfusions prior to and/or after administration of the genetically modified cells. In any of the methods described herein, after administration of the composition to the subject, the need for additional therapies such as a bone marrow transplant, blood component, iron chelation, and/or therapy PRBC transfusions in the subject are reduced or eliminated. In some embodiments, the need for additional therapies in the subject is reduced or eliminated within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more days after administration of the genetically modified cells.

[0120] In some of the methods or uses described herein, the subject has a confirmed diagnosis of sickle cell disease (SCD). In some embodiments, the subject has a confirmed diagnosis of severe SCD.

[0121] Disclosed herein are methods of treating a SCD in a subject in need thereof by administering (e.g., by infusion) a genetically modified cell in which BCL11A is inactivated in the cell to the subject such that HbF production in the subject is increased and one or more clinical symptoms of sickle cell disease are decreased.

[0122] The subjects with SCD that are treated may exhibit one or more of the following: (1) a change from baseline of clinical laboratory hemoglobin fractions (adult hemoglobin, HbA and fetal hemoglobin, HbF) in grams/dL plasma and/or percent HbF of total Hb; (2) alteration (e.g., to or near normal levels) of SCD-related disease biomarkers such biomarkers of iron metabolism; and/or levels of erythropoietin, haptoglobin and/or hepcidin; (3) reduction or elimination of symptoms in the subject associated with iron overload associated with baseline transfusion therapy; (4) reduction or elimination of the need for blood product infusions, including PRBC transfusions, platelet infusions, I VIG, plasma transfusion and/or granulocyte transfusion; and/or (5) a change from baseline (pre-treatment levels) in the number and percent of F cells.

[0123] In some applications, provided herein is a method of improving or maintaining (slowing the decline) of SCD-related disease biomarkers in a human subject having sickle cell disease as compared with a subject that has not been treated with the methods and compositions of the invention. In other applications, provided herein is a method of decreasing the need (dose level or frequency) for PRBC or other blood product infusions in a subject with SCD as compared with the subject prior to treatment with the methods and compositions of the invention. In yet another aspect, provided herein is a method of reducing iron overload in a patient with sickle cell disease that occurs from chronic blood product infusions.

[0124] In some embodiments, a subject with SCD is assessed for improvement in SCD. In some embodiments, the improvement in SCD is assessed by the decrease, amelioration, or elimination of one or more clinical symptoms of SCD. In some embodiments, the subject with SCD is assessed using a health-related quality of life (HRQoL) survey. In some embodiments, the subject with SCD is assessed using the Patient-Reported Outcomes Measurement Information System 57 (PROMIS-57). PROMIS-57 is a collection of 8-item short forms assessing physical and mental well-being in patients with SCD. Exemplary PROMIS measures include pain impact, pain behavior, physical functioning, anxiety, depression, fatigue, satisfaction with discretionary social activities, satisfaction with social roles, sleep disturbance, and sleep-related impairment. See, e.g., Keller S etal. (2017) Health Qua! Life Outcomes. 15(1):117; DeWalt, et al. (2007) Med Care. 45(5 Suppl 1):S12— 21. In the clinical study disclosed herein, the PROMIS-57 survey may be administered at the Screening visit, Week 26, Week 52, Week 78. End of Study Visit, and Eady Termination Visit. [0125] In some embodiments, a subject with SCD can be assessed using the Adult Sickle Cell Quality of Life Measurement System (ASCQ-Me). Keller SD, et al. (2014). Health Qual Life Outcomes. 22:125.

[0126] In some embodiments, a subject with SCD can be assessed using the Karnofsky Performance Scale. This is a simple, widely-accepted tool for evaluating functional impairment in patients. Each subject will be evaluated and scored at the specified visit using the Karnofsky Performance Status Scale Definitions Rating Criteria. See, e.g., Canver & Orkin (2016). Blood. 127(21):2536-2545.

Nucleases targeting BCL11A

[0127] Disclosed herein are compositions comprising one or more mRNAs encoding one or more zinc finger proteins (ZFNs) that cleave an endogenous BCL11A sequence e.g., an endogenous BCL11A enhancer sequence). In some embodiments, the one or more mRNAs comprise SB-mRENH1 mRNAs and/or SB-mRENH2 mRNAs (SEQ ID NO:15 and SEQ ID NO:16, respectively). See U.S. Patent No. 10,563,184; U.S. Patent Publication No. 2018/0087072. Also disclosed are pharmaceutical compositions comprising one or more of the same or different mRNAs, including compositions comprising SB-mRENH1 and SB-mRENH2 mRNAs.

[0128] In some embodiments, the target sites for the ZFNs disclosed herein are within a BCL11A gene. See, e.g., U.S. Patent Nos. 10,563,184; 9,963,715; 9,650,648; U.S. Patent Publication Nos. 2015/0132269; 2018/0111975; and 2019/0177709.

[0129] BCL11A is a transcription factor that is active in both neurological development and hematopoiesis. Genome-wide association and functional follow-up studies in cell and animal models have shown that BCL11A is an important silencer of HbF expression. In a seminal study involving a humanized mouse model of SCD, genetically engineered BCL11 A disruption in erythroid cells led to an absence of hemoglobin switching, maintenance of high levels of HbF, and improvements in hematologic and pathologic characteristics of SCD. (Xu et al. (2011) Science 334(6058): 993-6).

[0130] Thus, inhibition of BCL11A appears to be a potentially effective strategy for treating P-globin disorders such as SCD in human subjects. However, targeting the BCL11A gene for therapeutic approaches poses challenges due to the crucial role of BCL11A in development and hematopoiesis (Brendel et al. (2016) J Clin Invest 126(10:3868-3878). An alternative strategy targets an erythroid-specific enhancer (ESE) element that is located in the second intron of the BCL11 A and that is required for BCL11A expression in erythroid cells but not in other lineages. The enhancer element was found to contain a common genetic variation associated with higher HbF levels (Bauer et al. (2013) Science 342(6155):253-7). It was hypothesized that modification of this erythroid-specific enhancer of the BCL11A gene could boost endogenous HbF levels in erythroid cells without deleterious effects on global BCL11A function (Hardison & Blobel (2013) Science 342(6155): 206-7).

[0131] Disclosed herein are genetically modified cells {e.g., red blood cell (RBC) precursor cell such as a CD34+ hematopoietic stem cell or erythroid precursor cell) comprising a genetic modification within an endogenous BCL11A enhancer sequence, such that the BCL11 A gene is inactivated in the cell. Also provided are cell populations comprising these genetically modified cells; genetically modified cells descended from therefrom; cell populations comprising the genetically modified cells and cells descended therefrom; and compositions comprising the genetically modified cells and/or cells descended therefrom. The cells, cell populations, and compositions described herein may be autologous (from the subject) and/or allogeneic cells. Furthermore, the genetically modified cells may include one or more additional genetic modifications, including but not limited to cells in which one or more selfmarkers or antigens are inactivated (knocked-out).

[0132] In some embodiments, the methods disclosed herein comprise a knock out of the BCL11A enhancer sequence in a cell to block the expression of the BCL11A protein. The methods and compositions of the invention also can be used in any circumstance wherein it is desired to knock out the BCL11A erythroid enhancer in a hematopoietic stem cell such that mature cells (e.g., RBCs) derived from these cells contain the therapeutic knockout. These stem cells can be differentiated in vitro or in vivo and may be derived from a universal donor type of cell which can be used for all subjects. Additionally, the cells may contain a transmembrane protein to traffic the cells in the body. Treatment can also comprise use of subject cells containing the therapeutic transgene where the cells are developed ex vivo and then introduced back into the subject. For example, HSC/PC containing a BCL11A erythroid enhancer knockout may be inserted into a subject via an autologous bone marrow transplant. [0133] Thus, described herein are methods for altering hemoglobin expression for the treatment and/or prevention of sickle cell disease. In some embodiments a ZFN pair comprising first and second (left and right) ZFNs, namely a 6-finger ZFN comprising a ZFP designated 63014 comprising the recognition helix regions as shown in Table 1 (e.g., encoded by mRNA SB-mRENH1) and a 5-finger ZFN comprising a ZFP designated 65722 comprising the recognition helix regions as shown in Table 1 (e.g., encoded by mRNA SB-mRENH2) is used for altering hemoglobin levels in an isolated cell or cell of a subject. In some embodiments, the ZFN pair binds to a 33-base pair (combined) target site in the erythroid- specific enhancer of the human BCL11A gene at location chr2:60, 495, 250-60, 495, 290 in the GRCh38/hg38 assembly of the human genome. In some embodiments, one mRNA encodes both ZFNs of the pair. Alternatively, separate mRNAs, each encoding one ZFN of the pair are employed. In some embodiments, the mRNA sequences are SEQ ID NO:15 and SEQ ID NO:16.

[0134] In some embodiments, the ZFN useful in the compositions and methods disclosed herein {e.g., a ZFN in which the members of the ZFN pair (left and right) ZFNs are delivered by two separate mRNAs) include mRNAs designated SB-mRENH1 and SB-mRENH2. In some embodiments, the ZFNs in the BCL11A-specific pair are delivered (e.g., to the HSC/PC) via electroporation, for example, wherein one AAV comprises the left ZFN (e.g., SB-mRENH1) and another comprises the right ZFN (e.g., SB-mRENH2).

[0135] In some embodiments, the cells are removed from the subject (autologous) and treated with nucleases that target a gene involved in the regulation of fetal hemoglobin (HbF) production. In some embodiments, the gene is a repressor of HbF production. In some embodiments, the gene is the BCL11A gene. In some embodiments, the nucleases target and cleave the erythroid-specific enhancer region of the BCL11A gene. In some embodiments, the nucleases are delivered to the cells as mRNAs. In some embodiments, the cleavage of the erythroid-specific enhancer region results in error-prone repair of the cleavage site by the cellular repair machinery such that a binding site for the erythroid transcription factor GATA1 (see Vierstra et al. (2015) Nat Methods 12(10):927-30; Canver et al. (2015) Nature 527(7577): 192-7) is disrupted. In some embodiments, the nucleases target the erythroid-specific enhancer region of the BCL11A gene such that it is not expressed in hematopoietic stem cells. Enhancer regions targeted may be within or outside the coding region including but not limited to +58, +55 and/or +62 regions within intron 2 of endogenous BCL11A, numbered in accordance with the distance in kilobases from the transcription start site of BCL11A, which enhancer regions are roughly 350 (+55); 550 (+58); and 350 (+62) nucleotides in length. See, e.g., Bauer et al. (2013) Science 343:253-257; U.S. Patent Nos. 9,963,715; 10,072,066; and U.S. Patent Publication Nos. 2015/0132269 and 2018/0362926.

[0136] In some embodiments, the cells (populations of cells and compositions comprising these cells and populations of cells) described herein are specifically genetically modified by the mRNA(s) at the BCL11A locus, including genetically modified cell populations (and compositions comprising these cells) in which less than 10% (0 to 10% of any value therebetween), preferably less than 5% (0 to 5% or any value therebetween), even more preferably less than 1 % of the cells (0 to 1% or any value therebetween) and even more preferably less than 0.5% (0 to 1 % or any value therebetween) of the genetically modified cells include genetic modifications made by the mRNA(s) outside the BCL11A locus (but may include additional modifications such as inactivation of HLA markers). In some embodiments, the nuclease is encoded by an mRNA and the mRNA optionally comprises elements for increasing transcriptional and translational efficiency.

[0137] In some embodiments, described herein is a composition comprising genetically modified cells specifically modified at the BCL11 A locus by the mRNA(s) as described herein, including in which less than 10% (0 to 10% or any value therebetween), preferably less than 5% (0 to 5% or any value therebetween), even more preferably less than 1% of the cells (0 to 1% or any value therebetween) and even more preferably less than 0.5% (0 to 1% or any value therebetween) of the genetically modified cells include genetic modifications made by the mRNA(s) outside the BCL11A locus (but may include additional modifications such as inactivation of HLA markers). In further embodiments, the polynucleotides encoding the zinc finger nuclease may comprise a left ZFN known as SB63014 (see, U.S. Patent No. 10,563,184 and U.S. Patent Publication No. 2018/0087072), encoded by a mRNA SB-mRENH1 . In some embodiments, the right ZFN is SB65722 (see, U.S. Patent No. 10,563,184 and U.S. Patent Publication No. 2018/0087072), encoded by a mRNA SB-mRENH2.

[0138] In some embodiments, the left and right (first and second) ZFNs of the ZFN are carried on the same vector and in other embodiments, the paired components of the ZFN are carried on different vectors. For example, two mRNAs vectors, one designated SB-mRENH1 mRNA (an mRNA encoding the ZFN comprising the ZFP designated 63014) and the other designated SB-mRENH2 mRNA (an mRNA encoding the ZFN comprising the ZFP designated 65722).

[0139] The examples disclosed herein relate to exemplary embodiments of the present disclosure in which the nuclease comprises a zinc finger nuclease (ZFN). It will be appreciated that this is for purposes of exemplification only and that other nucleases or nuclease systems can be used, for instance homing endonucleases (meganucleases) with engineered DNA-binding domains and/or fusions of naturally occurring of engineered homing endonucleases (meganucleases) DNA-binding domains and heterologous cleavage domains and/or a CRISPR/Cas system comprising an engineered single guide RNA.

Cells

[0140] In one aspect, described herein are host cells, including isolated hematopoietic stem cells (HSPC such as CD34+), comprising the ZFNs and/or polynucleotides {e.g., mRNAs) as described herein. Cells may be isolated from healthy subjects or, alternatively, are autologous cells obtained from a subject with the condition to be treated {e.g., SCD) and purified using standard techniques. The ZFNs genetically modify the cells via insertions and/or deletions following cleavage. Subsequently, expanded (cultured) cells may no longer include the ZFNs (or polynucleotides encoding these ZFNs) but maintain the genetic modifications in culture e.g., insertions and/or deletions within BCL11a). Genetically modified cells as described herein exhibit different ratios of globin (a-, p- and y-globin levels) as compared to untreated (non-genetically modified) cells. In some embodiments, the ratio of y-globin to - globin and of y-globin to a-globin is increased about 2 to 5 or more-fold, including 3 to 4-fold as compared to untreated (untransfected) HSPCs. Furthermore, the genetically modified cells described herein differentiate into all hematopoietic lineages, including erythroid progenitors (CFLI-E and BFLI-E), granulocyte/macrophage progenitors (CFU-G/M/GM), and multipotential progenitors (CFU-GEMM) and exhibit normal karyotypes and morphology, which is indicative of a reconstitution of hematopoiesis.

[0141] Also provided herein are genetically modified cells, for example, HSC/PC comprising a targeted knockout of the BCL11 A erythroid enhancer. The knockout is created by treating harvested HSC/PC with mRNAs encoding the right and left ZFN partners which when translated, will result in an active ZFN. The ZFN cleaves the BCL11A erythroid enhancer such that a double strand break in the DNA occurs. The cellular machinery repairs the double strand break using error-prone non-homologous end joining (NHEJ) which results in the insertion and deletion of nucleotides (indels) around the cleavage site.

[0142] Both autologous {e.g., subject-derived) and allogenic (healthy donor derived) HSC/PC can be used in the performance of the method.

[0143] The cells as described herein are useful in cell therapy for treating and/or preventing sickle cell disease in a subject with the disorder. In the case of modified stem cells, after infusion into the subject, in vivo differentiation of these precursors into cells expressing the functional protein (from the inserted donor) also occurs.

[0144] Pharmaceutical compositions comprising the cells as described herein are also provided. In addition, the cells may be cryopreserved prior to administration to a subject.

[0145] The cell populations (and compositions) described herein comprise genetically modified cells specifically at the BCL11A locus, including genetically modified cell populations in which less than 10% (0 to 10% of any value therebetween), preferably less than 5% (0 to 5% or any value therebetween), even more preferably less than 1% of the cells (0 to 1% or any value therebetween) and even more preferably less than 0.5% (0 to 1% or any value therebetween) of the cells include genetic modifications outside the BCL11A locus (but may include additional modifications such as inactivation of HLA markers).

[0146] The genetically modified cells may be stem cells (e.g., CD34+ HSC/PC, ST-400) and may be autologous or allogeneic (e.g., isolated from healthy donors) and the allogeneic cells may be further modified (e.g., in addition to BCL11A inactivation), for example to remove one or more self-antigens e.g., HLA complexes) to from the allogeneic cells. See, e.g., U.S. Patent Nos. 8,945,868; 10,072,062; U.S. Patent Publication No. 2018/0362926. Autologous cells may be mobilized in the subject prior to modification ex vivo by treating the subject with one or more doses of plerixafor and the mobilized cells are harvested by one or more apheresis cycles. Optionally, at least about 25 x 10⁶ CD34+ HSPCs/kg are mobilized in the subject. The cells may be genetically modified to inactivate BCL11A using one or more nucleases, for example wherein the nucleases are introduced into the cell as mRNAs as disclosed herein (SEQ ID NO:15 and SEQ ID NO:16). Following ex vivo genetic modification, the cells may be evaluated for insertions and/or deletions within BCL11A.

[0147] Isolated cells and isolated populations of cells comprising one or more mRNAs and/or one or more pharmaceutical compositions comprising these mRNAs are also provided. Also described are compositions comprising genetically modified cells and cells descended therefrom, including, but not limited to, progeny of the genetically modified cells. The genetically modified progeny cells may be obtained by in vitro methods (culture of the genetically modified cells) and/or in vivo following administration of the genetically modified cells to a subject. Thus, the genetically modified progeny cells may include fully or partially differentiated progeny descended from the genetically modified cells. In some embodiments, the genetically modified cell compositions comprise genetically modified hematopoietic stem cells (also referred to as hematopoietic progenitor stem cells (HPSC) or hematopoietic stem cell/precursor cells (HSC/PC)) and/or genetically modified cells descended or produced (cultured) therefrom, including genetically modified cells in which the BCL11A sequence is cleaved and hemoglobin {e.g., HbF and/or HbA) levels in the cells are increased e.g., 3 to 4- fold or more) as compared to cells which are not genetically modified. Some, all or none of the genetically modified cells of the cell populations and compositions of cells described herein may comprise one or more mRNAs and/or pharmaceutical compositions comprising these mRNAs. Thus, described herein are cells, cell populations and compositions comprising these cells, which cells, cell populations and compositions comprise genetically modified cells comprising the mRNAs described herein and cells descended therefrom. The cells, cell populations and compositions comprising these cells and cell populations may comprise autologous and/or allogeneic cells.

[0148] Pharmaceutical compositions comprising genetically modified cells {e.g., erythroid progenitor cells such as HPSCs that exhibit increased globin expression as compared to unmodified cells) as described herein are also provided.

[0149] Methods of manufacturing (making) genetically modified isolated cells (or cell populations or compositions comprising genetically modified cells and cells descended therefrom) are also provided, including methods of making genetically modified populations of cells in which a BCL11 A sequence (e.g., enhancer sequence) is genetically modified such that hemoglobin {e.g., HbF and/or HbA) levels in the genetically modified cells are increased as compared to unmodified cells e.g., 2 or more fold). In some embodiments, the methods comprising administering one or more mRNAs (or pharmaceutical compositions comprising the one or more mRNAs) as described herein to the cell e.g., via transfection). The cells may be autologous and/or allogeneic and may be HSPCs. In some embodiments, the methods further comprise culturing the genetically modified cells to produce a composition comprising a population of genetically modified cells {e.g., HPSC cells) and/or genetically modified cells descended therefrom {e.g., other erythroid progenitor cells and/or mature erythroid cells such as RBCs) exhibiting increased globin production. The compositions may comprise genetically modified cells comprising the mRNAs and/or genetically modified cells descended from such cells that no longer comprise the mRNAs but maintain the genetic modification (BCL11A- specific modifications).

[0150] Pharmaceutical compositions comprising genetically modified cell populations and/or cells descended therefrom are also provided.

[0151] In some embodiments, the methods and compositions disclosed herein relate to treating a subject with cells that have been modified ex vivo. In some embodiments, the cells are isolated from the subject, modified ex vivo, and then returned to the subject. In other embodiments, the cells are isolated from healthy donors, modified ex vivo, and then used to treat the subject. In further embodiments, the cells isolated from healthy donors are further modified ex vivo to remove self-markers {e.g., HLA complexes) to avoid rejection of the cells by the subject. In some embodiments, the cells isolated are stem cells. In further embodiments, the stem cells are hematopoietic stem cell/progenitor cells {e.g., CD34+ HSC/PC). In some embodiments, the CD34+ HSC/PC are mobilized in each subject by treatment with one or more doses of plerixafor. In some embodiments, the dose of plerixafor used is about 240 pg/kg/day. In further embodiments, the mobilized cells are harvested by one or more apheresis cycles.

[0152] Mobilized human CD34+ HSPCs may be collected by apheresis from healthy or SCD subjects and purified prior to administration of (transfection with) one or more mRNAs (or pharmaceutical compositions comprising the one or more mRNAs) as described herein. In some embodiments, the purified HSPCs are transfected with ZFN mRNAs SBmRENHI (SEQ ID NO:15) and SBmRENH2 (SEQ ID NO:16) to manufacture SAR445136.

[0153] Transfected genetically modified CD34+ HSPCs (SAR445136) may be cultured, harvested and/or frozen for use. After harvesting, compositions comprising genetically modified cells (at least 50%, preferably at least 70% or more, even more preferably at least 75-80% or more of the cells are genetically modified following mRNA administration, preferably specifically modified at the BCL11A enhancer sequence as compared to other genetic loci) as described herein may include HSPCs as well as cells descended therefrom, for instance HSPC differentiated into all hematopoietic lineages, including erythroid progenitors (CFLI-E and BFLI-E), granulocyte/macrophage progenitors (CFU-G/M/GM), and multi-potential progenitors (CFU-GEMM). In some embodiments, some, none or all of the genetically modified cells of the composition (population) of cells comprise one or more of mRNAs.

[0154] In another aspect, provided herein is an article of manufacture comprising a package (for example, a bag) comprising compositions comprising genetically modified autologous HSC/PC as described herein. The article of manufacture {e.g., bag) may be formulated for frozen storage, for example in CryoStor® CS-10 cryomedia (SigmaAldrich) containing 10% DMSO. Each bag can contain any concentration of cells. In some embodiments, each bag contains approximately 1 .0 - 2.0 x 10⁸ cells per bag at a concentration of approximately 1 x 10⁷ cells/mL.

[0155] In some embodiments, disclosed herein are compositions comprising (1) BCL11A gene ESE-modified CD34+ HSPC at 10 x 10⁶ cells/mL; and (2) 1 mL of cryopreservation buffer containing 10% dimethyl sulfoxide (DMSO). In some embodiments, the cryopreservation buffer is CryoStor® CS10. In some embodiments, the composition is SAR445136.

Delivery

[0156] The ex vivo delivery of nucleases, polynucleotides encoding these nucleases, donor polynucleotides and compositions comprising the proteins and/or polynucleotides described herein may be delivered to the harvested HSC/PC by any suitable means.

[0157] Methods of delivering nucleases as described herein are described, for example, in U.S. Patent Nos. 6,453,242; 6,503,717; 6,534,261 ; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, the disclosures of all of which are incorporated by reference herein in their entireties.

[0158] Nucleases and/or donor constructs as described herein may also be delivered using vectors containing sequences encoding one or more of the zinc finger, TAL-effector domain and/or Cas protein(s). Any vector systems may be used including, but not limited to, plasmid vectors, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirus vectors and adeno-associated virus vectors, etc. See, also, U.S. Patent Nos. 6,534,261 ; 6,607,882; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, incorporated by reference herein in their entireties.

[0159] Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding nucleases and donor constructs in cells (e.g., mammalian cells) and target tissues. Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see, Anderson (1992) Science 256:808-813; Nabel & Feigner (1993) TIBTECH 11 :211-217; Mitani & Caskey (1993) TIBTECH 11 :162-166; Dillon (1993) TIBTECH 11:167- 175; Miller (1992) Nature 357:455-460; Van Brunt (1988) Biotechnology 6(10):1149-1154; Vigne (1995) Restorative Neurology and Neuroscience 8:35-36; Kremer & Perricaudet (1995) British Medical Bulletin 51(1):31-44; Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Bohm (eds.) (1995); and Yu et al. (1994) Gene Therapy 1:13-26.

[0160] Methods of non-viral delivery of nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids.

[0161] Additional exemplary nucleic acid delivery systems include those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Maryland), BTX Molecular Delivery Systems (Holliston, MA) and Copernicus Therapeutics Inc, (see for example U.S. Patent No. 6,008,336). Lipofection is described in e.g., U.S. Patent Nos. 5,049,386; 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, International Patent Publication Nos. WO 91/17424, WO 91/16024.

[0162] The preparation of lipidmucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal (1995) Science 270:404-410; Blaese et al. (1995) Cancer Gene Ther. 2:291-297; Behr et al. (1994) Bioconjugate Chem. 5:382-389; Remy et al. (1994) Bioconjugate Chem. 5:647-654; Gao et al. (1995) Gene Therapy 2:710-722; Ahmad et al. (1992) Cancer Res. 52:4817-4820; U.S. Patent Nos. 4,186,183; 4,217,344; 4,235,871; 4,261 ,975; 4,485,054; 4,501,728; 4,774,085; 4,837,028; and 4,946,787).

[0163] Additional methods of delivery include the use of packaging the nucleic acids to be delivered into EnGenelC delivery vehicles (EDVs). These EDVs are specifically delivered to target tissues using bispecific antibodies where one arm of the antibody has specificity for the target tissue and the other has specificity for the EDV. The antibody brings the EDVs to the target cell surface and then the EDV is brought into the cell by endocytosis. Once in the cell, the contents are released (see, MacDiarmid et al. (2009) Nature Biotechnology 27(7):643). [0164] The use of RNA or DNA viral based systems for the delivery of nucleic acids encoding engineered ZFPs take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be used to treat cells in vitro and the modified cells are administered to subjects (ex vivo). Conventional viral based systems for the delivery of ZFPs include, but are not limited to, retroviral, lentivirus, adenoviral, adeno-associated, vaccinia and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been measured in many different cell types and target tissues.

[0165] Recombinant adeno-associated virus vectors (rAAV) are a promising alternative gene delivery system based on the defective and nonpathogenic parvovirus adeno-associated type 2 virus. All vectors are derived from a plasmid that retains only the AAV 145 bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genomes of the transduced cell are key features for this vector system. (Wagner et al. (1998) Lancet 351(9117):1702-3; Kearns et al. (1996) Gene Ther. 9:748-55). Other AAV serotypes, including by non-limiting example, AAV1 , AAV3, AAV4, AAV5, AAV6, AAV8, AAV 8.2, AAV9 and AAV rh10 and pseudotyped AAV such as AAV2/8, AAV2/5 and AAV2/6 can also be used in accordance with the present invention. In some embodiments, AAV serotypes that are capable of crossing the blood brain barrier are used.

[0166] Replication-deficient recombinant adenoviral vectors (Ad) can be produced at high titer and readily infect a number of different cell types. Most adenovirus vectors are engineered such that a transgene replaces the Ad E1a, E1b, and/or E3 genes; subsequently the replication defective vector is propagated in human 293 cells that supply deleted gene function in trans. Ad vectors can transduce multiple types of tissues in vivo, including nondividing, differentiated cells such as those found in liver, kidney and muscle. Conventional Ad vectors have a large carrying capacity. An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for anti-tumor immunization with intramuscular injection (Sterman et al. (1998) Hum. Gene Ther. 7:1083-9). Additional examples of the use of adenovirus vectors for gene transfer in clinical trials include Rosenecker et al. (1996) Infection 24(1):5-10; Sterman et al. (1998) Hum. Gene Ther. 9(7): 1083-1089; Welsh et al. (1995) Hum. Gene Ther. 2:205-18; Alvarez et al. (1997) Hum. Gene Ther. 5:597-613; Topf et al. (1998) Gene Ther. 5:507-513; Sterman et al. (1998) Hum. Gene Ther. 7:1083-1089.

[0167] Packaging cells are used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and i 2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by a producer cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host (if applicable), other viral sequences being replaced by an expression cassette encoding the protein to be expressed. The missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess inverted terminal repeat (ITR) sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g. , heat treatment to which adenovirus is more sensitive than AAV.

[0168] Compositions comprising genetically modified cells as described herein may be delivered to a subject in any suitable manner, including by infusion. Prior to administration of composition comprising the genetically modified cells, the subject may be treated with (administered) one or more myeloablative condition agents one or more times, for example, busulfan administered: intravenously (IV) at between about 0.5 to 5 mg/kg for one or more times; IV at about 3.2 mg/kg/day; IV via central venous catheter for 4 days total dose of about 12.8 mg/kg prior to infusion on Days -6 through -3 before infusion of the composition comprising the genetically modified cells on Day 0; or IV once daily or every 6 hours.

[0169] Any dose of genetically modified cells can be used, for example, between about 3 x 10⁶ cells/kg and about 20 x 10⁶ cells/kg (e.g., where the cells are formulated with approximately 1.0- 2. O x 10⁸ cells per bag at a concentration of approximately 1 x 10⁷ cells/mL). [0170] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions available, as described below (see, e.g., Remington’s Pharmaceutical Sciences, 17th ed., 1989).

[0171] Formulations for both ex vivo and in vivo administrations include suspensions in liquid or emulsified liquids. The active ingredients often are mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances, such as, wetting or emulsifying agents, pH buffering agents, stabilizing agents or other reagents that enhance the effectiveness of the pharmaceutical composition.

[0172] In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.

EXAMPLES

[0173] The foregoing description of the specific embodiments will so fully reveal the general nature of the disclosure that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

Example 1 : ZFN design

[0174] The ZFN pair is made up of a 6-finger ZFN (encoded by mRNA SB-mRENH1) and a 5-finger ZFN (encoded by mRNA SB-mRENH2) that binds to a 33 base pair (combined) target site in the erythroid-specific enhancer of the human BCL11A gene at location chr2:60, 495, 250-60, 495, 290 in the GRCh38/hg38 assembly of the human genome. The preparation of the ZFN and polynucleotides encoding them is as follows: The SB- mRENHIand SB-mRENH2 mRNAs are produced in vitro by methods known in the art. The mRNAs comprise sequences encoding the ZFN partners, and also comprise features such as nuclear localization sequences and peptides. Table 1 shows the helices associated with each partner ZFN (see U.S. Patent No. 10,563,184; U.S. Patent Publication No. 2018/0087072):

Table 1 : ZFN design

[0175] The complete nucleotide sequence for the SB-mRENH1 mRNA (1725 nucleotides) is shown below (SEQ ID NO: 15): gggagacaagcuuugaauuacaagcuugcuuguucuuuuugcagaagcucagaauaaacgcucaacuuuggcagaucgaauu cgccauggacuacaaagaccaugacggugauuauaaagaucaugacaucgauuacaaggaugacgaugacaagauggccccca agaagaagaggaaggucggcauccacgggguacccgccgcuauggcugagaggcccuuccagugucgaaucugcaugcagaac uucagugaccaguccaaccugcgcgcccacauccgcacccacaccggcgagaagccuuuugccugugacauuugugggaggaaa uuugcccgcaacuucucccugaccaugcauaccaagauacacacgggcagccaaaagcccuuccagugucgaaucugcaugcag aacuucaguuccaccggcaaccugaccaaccacauccgcacccacaccggcgagaagccuuuugccugugacauuugugggagg aaauuugccaccuccggcucccugacccgccauaccaagauacacacgcacccgcgcgccccgaucccgaagcccuuccaguguc gaaucugcaugcagaacuucagugaccaguccaaccugcgcgcccacauccgcacccacaccggcgagaagccuuuugccugug acauuugugggaggaaauuugccgcccaguguugucuguuccaccauaccaagauacaccugcggggauccaucagcagagcc agaccacugaacccgcacccggagcuggaggagaagaaguccgagcugcggcacaagcugaaguacgugccccacgaguacau cgagcugaucgagaucgccaggaacagcacccaggaccgcauccuggagaugaaggugauggaguucuucaugaagguguacg gcuacaggggaaagcaccugggcggaagcagaaagccugacggcgccaucuauacagugggcagccccaucgauuacggcgug aucguggacacaaaggccuacagcggcggcuacaaucugccuaucggccaggccgacgagauggagagauacguggaggagaa ccagacccgggauaagcaccucaaccccaacgagugguggaagguguacccuagcagcgugaccgaguucaaguuccuguucg ugagcggccacuucaagggcaacuacaaggcccagcugaccaggcugaaccacaucaccaacugcaauggcgccgugcugagcg uggaggagcugcugaucggcggcgagaugaucaaagccggcacccugacacuggaggaggugcggcgcaaguucaacaacggc gagaucaacuucagaucuugauaacucgagucuagaagcucgcuuucuugcuguccaauuucuauuaaagguuccuuuguucc cuaaguccaacuacuaaacugggggauauuaugaagggccuugagcaucuggauucugccuaauaaaaaacauuuauuuuca uugcugcgcuagaagcucgcuuucuugcuguccaauuucuauuaaagguuccuuuguucccuaaguccaacuacuaaacuggg ggauauuaugaagggccuugagcaucuggauucugccuaauaaaaaacauuuauuuucauugcugcgggacauucuuaauua aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaacuag

[0176] The complete nucleotide sequence for SB-mRENH2 mRNA (1680 nucleotides) is shown below (SEQ ID NO: 16): gggagacaagcuugaauacaagcuugcuuguucuuuuugcagaagcucagaauaaacgcucaacuuuggcagaucgaauucg ccuagagaucuggcggcggagagggcagaggaagucuucuaaccugcggugacguggaggagaaucccggcccuaggaccaug gacuacaaagaccaugacggugauuauaaagaucaugacaucgauuacaaggaugacgaugacaagauggcccccaagaaga agaggaaggucggcauucaugggguacccgccgcuauggcugagaggcccuuccagugucgaaucugcaugcagaaguuugcc cgcaacgaccaccgcaccacccauaccaagauacacacgggcgagaagcccuuccagugucgaaucugcaugcagaacuucagu cagaaggcccaccugauccgccacauccgcacccacaccggcgagaagccuuuugccugugacauuugugggaggaaauuugcc cagaagggcacccugggcgagcauaccaagauacacacgggaucucagaagcccuuccagugucgaaucugcaugcagaacuu cagucgcggccgcgaccugucccgccacauccgcacccacaccggcgagaagccuuuugccugugacauuugugggaggaaauu ugcccgccgcgacaaccugcacucccauaccaagauacaccugcggggaucccagcuggugaagagcgagcuggaggagaaga aguccgagcugcggcacaagcugaaguacgugccccacgaguacaucgagcugaucgagaucgccaggaacagcacccaggac cgcauccuggagaugaaggugauggaguucuucaugaagguguacggcuacaggggaaagcaccugggcggaagcagaaagc cugacggcgccaucuauacagugggcagccccaucgauuacggcgugaucguggacacaaaggccuacagcggcggcuacaau cugccuaucggccaggccgacgagaugcagagauacgugaaggagaaccagacccggaauaagcacaucaaccccaacgagug guggaagguguacccuagcagcgugaccgaguucaaguuccuguucgugagcggccacuucagcggcaacuacaaggcccagc ugaccaggcugaaccgcaaaaccaacugcaauggcgccgugcugagcguggaggagcugcugaucggcggcgagaugaucaaa gccggcacccugacacuggaggaggugcggcgcaaguucaacaacggcgagaucaacuucugauaacucgagucuagaagcuc gcuuucuugcuguccaauuucuauuaaagguuccuuuguucccuaaguccaacuacuaaacugggggauauuaugaagggcc uugagcaucuggauucugccuaauaaaaaacauuuauuuucauugcugcgcuagaagcucgcuuucuugcuguccaauuucu auuaaagguuccuuuguucccuaaguccaacuacuaaacugggggauauuaugaagggccuugagcaucuggauucugccua auaaaaaacauuuauuuucauugcugcgggacauucuuaauuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaaacuag

Example 2: Clinical study of zinc finger nuclease-modified autologous CD34+ hematopoietic stem/progenitor cells for sickle cell disease

[0177] A study was undertaken in humans to test the safety and tolerability of using modified hematopoietic stem/progenitor cells (HSPCs) to treat sickle cell disease (SCD).

[0178] Sickle cell disease (SCD) is an inherited condition caused by pathologic variants in both alleles of the p-globin gene, affecting approximately 100,000 patients in the US. Strouse J. (2016) Handbook Clin Neurol. 138:311-24. Elevated fetal hemoglobin (HbF) levels have been shown to ameliorate symptoms and improve survival in SCD patients. Hebert N, et al. (2020). Am J Hematol. 95:1235-1245.

[0179] SAR445136 is a novel therapeutic product which is composed of a patient’s own

(autologous) hematopoietic stem/progenitor cells (HSPC) in which the BCL11A gene is modified to enable production of disease modifying levels of HbF. The target for gene editing is an erythroid-specific enhancer (ESE) in the BCL11A gene. Mutations at the ESE locus down-regulate BCL11A expression specifically in erythroid precursors, resulting in elevated levels of HbF synthesis. Following myeloablation of the patient’s bone marrow, the genetically modified HSPC are returned to the donor to repopulate the hematopoietic lineages including erythrocytes. As an autologous bone marrow transplant, there is minimal risk of stem cell rejection or graft-versus-host disease. Newly synthesized RBCs derived from the edited SAR445136 stem cell population may have an HbF level that is protective against the pathophysiologic changes underlying SCD.

Primary Endpoints

[0180] The primary objective of the study is to evaluate the safety and tolerability of SAR445136 in subjects with severe SCD. The primary safety and tolerability endpoints are as follows: (1) Survival at post-transplantation Day 100, Week 52, and Week 104 (Last Study Visit); (2) Successful engraftment as defined in the protocol; and (3) occurrence of adverse events (AEs) and serious adverse events (SAEs).

Secondary Endpoints

[0181] The secondary objectives of the study are to evaluate the success and kinetics of SAR445136 stem cell collection, manufacturing, and engraftment and SAR445136 efficacy. The endpoints related to the success and kinetics of SAR445136 stem cell collection, manufacturing, and engraftment are as follows: (1) CD34+ HSPC yield from plerixafor stem cell mobilization; (2) proportion of subjects who achieve sufficient stem cell mobilization for rescue aliquot and SAR445136 production; (3) yield of ZFN-edited CD34+ HSPC (investigational product); (4) time to initial neutrophil recovery following SAR445136 infusion (first of 3 consecutive days with an absolute neutrophil count [ANC] >500/pL); (5) time to platelet recovery following SAR445136 infusion (first of 3 consecutive measurements with a platelet count >50,000/pL at least 1 week [7 days] following last platelet transfusion); (6) maintenance of ANC >500/pL and platelet count >50,000/pL to last patient visit; and (7) reconstitution of immune function post-SAR445136 transplantation, including but not limited to lymphocyte counts and immunoglobulin levels.

[0182] The endpoints related to efficacy include change from baseline in blood markers, including peripheral blood HbF, %F cells, and HbS; peripheral blood total Hb concentration; and markers of hemolysis including reticulocyte count, lactate dehydrogenase (LDH), haptoglobin, and serum bilirubin. Other efficacy endpoints include change from baseline in clinical assessments, including frequency and severity of SCD-related clinical events (e.g., VOC, pain episodes, etc.); quality of life (QoL) measures including fatigue; and RBC transfusion requirements, such as annualized number and total volume.

Exploratory Endpoints

[0183] Exploratory objectives of the study relate to changes in clinical parameters, features of the edited cell product, and biomarkers of disease. The exploratory endpoints may include the following: (1) changes from pretransplant baseline in clinical laboratory tests, physical examination, and vital sign parameters (2); assessment of on-target BCL11A gene modification in drug product and its correlation with HbF response (3) assessment of on-target BCL11A gene modification in peripheral blood white blood cell (WBC) populations and bone marrow erythroid progenitors from SAR445136 transplantation through the last study visit (4) assessment of off-target editing profile that may include karyotyping in SAR445136 retains and post-infusion peripheral blood and bone marrow samples up to the last study visit; (5) assessment of subject DNA sequence variation that may be associated with study safety or efficacy outcomes; and/or (6) changes from baseline through last study visit in SCD-related hematologic disease biomarkers in blood cells, plasma, or serum; end-organ function/imaging, including but not limited to central nervous system (CNS), lung, and kidney, as well as neuropsychological and neurocognitive function.

Inclusion Criteria

[0184] Diagnosis and inclusion criteria for the study included eight adult subjects with a diagnosis of severe SCD, 18 to 40 years of age at screening, who were willing and able to undergo autologous HSPC transplantation. [0185] The male and female participating subjects were between the ages of 18 and 40 years old, inclusive, at the time of informed consent with clinical diagnosis of SCD and confirmed molecular genetic diagnosis of an HbSS or HbSp⁰ genotype. Subjects completed age appropriate cancer screening, used double- barrier method of contraception through the entire study period, and consented to receive blood transfusions.

[0186] The subjects had a diagnosis of severe SCD. Severe SCD was defined as having 1 or more of the following manifestations consistent with Bone Marrow T ransplant Clinical T rial Network (BMT CTN) guidelines: (1) clinically significant neurologic event (stroke) or any neurological deficit lasting >24 hours; (2) history of 2 or more episodes of ACS in the 2-year period preceding informed consent despite adequate supportive care measures (i.e. , asthma therapy); (3) three or more pain crises per year in the 2-year period preceding informed consent (requiring IV pain management in the outpatient or inpatient hospital setting); (4) history of two or more episodes of priapism with subject seeking medical care in the 2-year period preceding informed consent; (5) administration of regular RBC transfusion therapy in the year preceding informed consent, defined as receiving 8 or more transfusions to prevent vaso-occlusive clinical complications (i.e., pain, stroke, or ACS); and/or (6) an echocardiographic finding of tricuspid valve regurgitant jet (TRJ) velocity >2.5 m/s.

[0187] The subjects were required to have adequate physiological function consistent with BMT CNT guidelines. For example, adequate physiological function was measured by (a) Karnofsky/Lansky Performance Scale score >60; (b) cardiac function - left ventricular ejection fraction (LVEF) >40% or left ventricular shortening fraction >26% by cardiac echocardiogram (ECHO) or by multiple gated acquisition (MLIGA) scan (c) pulmonary function - pulse oximetry with a baseline oxygen (02) saturation of >85% and diffusing capacity for carbon monoxide (DLCO) >50% (corrected for hemoglobin); and (d) hepatic function - alanine aminotransferase (ALT) and aspartate aminotransferase (AST) <5* the upper limit of normal as per local laboratory: serum conjugated (direct) bilirubin <3* the upper limit of normal for age as per local laboratory. If direct bilirubin is >0.5 mg/dL, subject must have either a liver magnetic resonance imaging (MRI) with a liver iron content (LIC) <10 mg/g dry weight, or a liver biopsy demonstrating absence of bridging fibrosis, liver cirrhosis, and active hepatitis in order to participate (results of liver biopsies obtained within the past 12 months are allowed). Subjects with hyperbilirubinemia as a consequence of hyperhemolysis or who experienced a sudden, profound change in the serum hemoglobin after an RBC transfusion were not excluded.

[0188] Lastly, the subjects had to be clinically stable and medically eligible to undergo stem cell mobilization and myeloablative hematopoietic stem cell transplantation (HSCT) based on institutional medical guidelines. Exclusion Criteria

[0189] The key exclusion criteria for subjects in the study were as follows: previous receipt of an autologous or allogeneic HSCT or solid organ transplantation; previous treatment with gene therapy; current enrollment in an interventional study or having received an investigational drug within 30 days of study enrollment; pregnant or breastfeeding female; female or male who plans to become pregnant or impregnate a partner, respectively, during the anticipated study period; known to have a y-globin allelic variant associated with clinically significant altered oxygen affinity; a diagnosis of hereditary persistence of fetal hemoglobin (HPFH) or HbF concentration >20% at screening; medical contraindication to use plerixafor, apheresis, or busulfan; ANC <1000 cells/pL; platelet count <100,000 cells/pL known history of platelet alloimmunization precluding ability to provide transfusion support, extensive RBC alloimmunization precluding ability to provide transfusion support; treatment with prohibited medications in the previous 30 days (e.g., prohibited medications are shown in Table A; see supplementary tables section below); clinically significant, active bacterial, viral, fungal, or parasitic infection (based on Investigator’s judgement); screening laboratory testing demonstrating a diagnosis of any of the following: (a) human immunodeficiency virus (HIV), active hepatitis B virus infection (HBV) defined as positive for hepatitis B surface antigen [HBsAg] and hepatitis B core antibody [HBcAb] followed by detectable HBV DNA to confirm, or (c) active hepatitis C virus infection (HCV) defined as positive for hepatitis C antibody followed by detectable HCV RNA (US Centers for Disease Control and Prevention); any major organ dysfunction involving brain, kidney, liver, lung, or heart, including but not limited to the following: (a) congestive heart failure New York Heart Association (NYHA) Class 3 or 4, or patient with a past history of congestive heart failure NYHA Class 3 or 4 in whom ECHO or MUGA scan performed within 3 months prior to Screening showed an LVEF of <40%; and/or (b) pulmonary hypertension on treatment; Fridericia’s corrected QT interval (QTcF) >500 ms based on screening electrocardiogram (ECG); history of significant bleeding disorder; current diagnosis of uncontrolled seizures; history of active malignancy in the past 5 years (nonmelanoma skin cancer or cervical cancer in situ permitted), any history of hematologic malignancy, or family history of a cancer predisposition syndrome without negative testing results in the study candidate; known allergy or hypersensitivity to plerixafor, busulfan, or investigational product excipients (human serum albumin, dimethyl sulfoxide (DMSO), and Dextran 40); and any other reason that, in the opinion of the Investigator and/or Sponsor, that would have rendered the subject unsuitable for participation in the study. Withdrawal Criteria

[0190] The key subject withdrawal criteria were as follows: request by the subject to withdrawal; request of the Investigator and/or Sponsor if she/he thinks the study is no longer in the best interest of the subject; pregnancy of subject prior to IV busulfan infusion; subject judged by the Investigator to be at significant risk of failing to comply with the provisions of the protocol as to cause harm to self or seriously interfere with the validity of the study results; subject’s drug product does not fulfill release criteria or is otherwise determined to be unsuitable to administer to the subject (e.g., due to manufacturer/shipping problems); and/or withdrawal request at the discretion of the institutional review board (IRB), Office of Human Research, regulatory authority (e.g., FDA), Investigator, and/or the Sponsor. If a subject withdrew consent or discontinued from the study post-study treatment, a conference between the Investigator and the Medical Monitor took place to ensure and document that the subject understood the importance of the study follow-up and that the study treatment could not be revised even if the subject dropped out of the study follow-up. If the subject agreed, a reduced follow-up testing schedule may have been arranged including telephone calls and clinical laboratory tests to assess AEs and clinical status for up to 104 weeks after infusion. Subjects continued to be followed for overall survival unless consent to do so was withdrawn.

Study Design

[0191] The study was performed on subjects with sickle cell disease. Briefly, eligible subjects underwent mobilization and apheresis with plerixafor 240 ug/kg/day for up to 3 days to collect autologous CD34+ HSPCs at a minimum of 10 x 1 o⁶ CD34+ HSPC/kg per apheresis cycle to achieve the minimum SAR445136 dose. Additional apheresis cycles were permitted to achieve the minimum cell dose and rescue aliquots. Autologous HSPCs were transfected ex vivo with ZFN mRNAs SB-mRENH1 and SB-mRENH2, which target the ESE region of the BCL11A locus to manufacture SAR445136.

[0192] Subjects received conditioning therapy with a myeloablative dose of intravenous (IV) busulfan before being infused with the modified HSPCs. A single IV infusion of 3 to 20 x 10⁶ CD34+ HSPC/kg was administered at least 72 hours after the final busulfan myeloablation dose. Subjects were monitored for stem cell engraftment and hematopoietic recovery, adverse events (AEs), clinical and laboratory markers of hemolysis, total Hb and HbF, percentage of F cells and sickle-cell related events post-SAR445136 infusion. Health-related quality of life (HRQoL) was assessed via the PROMIS-57 survey at four timepoints (screening, Week 26, Week 52, early termination visit). The sequence and timing of events in the study are shown in FIG. 3. [0193] Schedule of screening, stem cell mobilization with plerixafor, apheresis, and conditioning with IV busulfan are shown in Table B (see supplementary tables section below). [0194] Administration of plerixafor and mobilization in each subject took between 4 to 7 days. Plerixafor was administered subcutaneously (SQ) at a recommended dose of 240 pg/kg/day. Plerixafor dosing, timing, or route of administration was modified or discontinued at the discretion of the Investigator based on the clinical status of the subject or if the WBC exceeded 100,000/pL.

[0195] The apheresis procedure required a temporary central IV catheter after one or more rounds of plerixafor administration. The minimum yield to secure sufficient stem cells for both the manufacturing of the investigational product and the rescue aliquot is approximately 10.0 x 10⁶ CD34+ HSPC/kg. A yield of 20 x 10⁶ CD34+ HSPC/kg may be required to achieve the target SAR445136 dose of 10 x 10⁶CD34+ HSPC/kg. Lower yields from a single apheresis cycle were acceptable but may trigger additional rounds of mobilization/apheresis. The timing of subsequent apheresis was at the discretion of the Investigator based on the subject’s clinical status. The time from stem cell harvest to edited stem cell infusion (SAR445136) was approximately 20 weeks.

[0196] An aliquot of unmanipulated cells from the apheresis was also collected and stored at the site if needed to secure a rescue treatment. The rescue aliquot portion comprised a minimum of 1.5 x 10⁶ CD34+ HSPC/kg. The remaining CD34+ HSPC were transfected ex vivo with ZFN mRNAs targeting the BCL11A locus to manufacture the investigational product SAR445136. The rescue aliquot was cryopreserved unmodified and stored at the study site for back-up infusion if the subject did not demonstrate evidence for engraftment by Day 42 post-SAR445136 infusion or developed subsequent graft failure with aplasia. In addition, the rescue treatment may have been administered at any time after SAR445136 infusion at the discretion of the investigator.

[0197] After removal and storage of the rescue treatment, the remainder of the subject’s mobilized and harvested cells were sent by courier to the GMP manufacturing facility. A CD34+ cell selection followed by transfection with ZFN mRNAs SB-mRENH1 and SB- mRENH2 to disrupt the erythroid-specific enhancer of the BCL11A gene was performed to generate the modified HSPC study drug. The modified HSPC were cryopreserved and stored until all the clinical protocol segments up to and including the Baseline visit procedures are completed and the subject is ready for infusion. The modified HSPC were cryopreserved in 50 mL CryoMACS® freezing bags (fill volume of approximately 10 to 20 mL; total cell count of approximately 1.0 x 10⁸ to 2.0 x 10⁸ cells) using a controlled rate freezer. Multiple freezing bags were used if cell yield exceeds the capacity of a single bag. Infusion bags were stored in vapor phase liquid nitrogen (at < -150^oC; with temperature monitoring) at the manufacturing facility until they were ready to be shipped to the clinical study center.

[0198] SAR445136 components include (1) BCL11A gene ESE-modified CD34+ HSPC at 10 x 10⁶ cells/mL, and (2) 1 mL of cryopreservation buffer (CryoStor® CS10) containing 10% dimethyl sulfoxide (DMSO).

[0199] After release of the modified HSPC for clinical use, the subjects proceeded with IV busulfan conditioning in a dedicated transplant unit for approximately 4 to 7 days total. Subjects received a myeloablative regimen of busulfan (3.2 mg/kg/day; IV via central venous catheter) for 4 days (total dose of 12.8 mg/kg, which is considered standard-of-care for autologous transplantation) on Days -6 through -3 before infusion of SAR445136 on Day 1 (72 hour rest period prior to infusion). After the first dose, the IV busulfan dose was adjusted based on pharmacokinetic (PK) sampling and study center practices to target an area under the curve (AUC) of 4,000-5,000 pmol*min for daily dosing. PK samples taken and analyzed after the first IV busulfan dose may have been used to adjust the third IV busulfan dose, and PK samples taken after the second IV busulfan dose may have been used to adjust the fourth IV busulfan dose. Seizure prophylaxis was required starting prior to and continuing at least 24 hours after the final dose of IV busulfan conditioning and iron-chelating agents were discontinued 2 weeks prior to IV busulfan.

[0200] Modified HSPC infusion: After myeloablative conditioning with intravenous busulfan, patients received the thawed CD34+ HSPCs (SAR445136) product by central venous catheter infusion. The frozen modified HSPC were thawed and infused, such that the thawing of the infusion bags would take approximately 2-5 mins/bag and the cells were within 40 minutes (from start of thaw to completion of infusion). The volume of frozen modified HSPC was determined by the subject’s weight. Vital signs (blood pressure, temperature, heart rate, respiratory rate and pulse oximetry) were monitored prior to infusion and afterwards.

[0201] Once given the study drug, the subjects were monitored for routine lab work. In addition, assessment of any adverse events and blood cells were assayed for gene modification. The tests, assessments, and events after SAR445136 infusion are shown in Table B. Tests, assessments, and events included evaluation of HbF levels, analysis of endocrine function, and MRIs performed to assess iron load. Kinetics and success of hematopoietic reconstitution, duration of hospitalization after conditioning, screening for potential development of hematological malignancies, overall function by Karnofsky performance score, efficiency of apheresis procedure, difference between % indels in SAR445136 product and indels detected in bone marrow and blood following SAR445136 infusion were also evaluated. Additional post-infusion assessments and their schedules are shown in Table C. [0202] An Adverse Event (AE) is any untoward medical occurrence in a subject administered a pharmaceutical product that does not necessarily have a causal relationship with this treatment. An AE can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease temporally associated with the use of a medicinal (investigational) product, whether or not related to the medicinal (investigational) product. Determination of whether an abnormal laboratory value, vital sign result, and/or ECG result meet the definition of an AE was made by the Investigator. Abnormal results are not considered AEs unless 1 or more of the following criteria are met: (1) the result meets the criteria for an SAE (2) the result requires the subject to receive specific corrective therapy or (3) the result is considered by the Investigator to be clinically significant. Myeloablation is intended through the use of the pretransplant conditioning regimen (busulfan at the full, myeloablative dose) and required for treatment efficacy. As such, hematologic abnormalities that are a direct consequence of the conditioning regimen are not considered AEs in this study and need not be reported as such unless the abnormality is of unanticipated severity or duration. Complications that are associated with these hematologic abnormalities (i.e. , fever, infection, and bleeding) must be reported as AEs.

[0203] A Serious Adverse Event (SAE) is any AE that results in any of the following outcomes: death, life-threatening threatening event (j.e., an event that places the subject at immediate risk of death); however, this does not include an event that, had it occurred in a more severe form, might have caused death, inpatient hospitalization or prolongation of existing hospitalization, persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions, congenital anomaly/birth defect in the offspring of an exposed subject, or a medically important event.

Results

[0204] Hb fractionation following SAR445136 infusion:

[0205] Eight (8) subjects have underwent mobilization and apheresis to date (September 22, 2021). Of these 8 subjects, 5 achieved successful target yields ranging from 3.4 to 13.8 x 10⁶ CD34+ HSPC/kg per apheresis day (mean: 6.49 x 10⁶ CD34+ HSPC/kg per apheresis day) in one apheresis cycle (4.45 to 10.9 x 10⁶ CD34+ HSPC/kg per2-day cycle). Two subjects failed to mobilize; one discontinued due to intercurrent cholangitis. One subject is scheduled for infusion. Baseline patient characteristics of the 4 subjects infused are shown in Table 5.

Table 5. Baseline Characteristics and Clinical History

[0206] Pre-apheresis peripheral blood WBC ranged from 23.2 to 36.9 x 10³/pL (mean: 28.7 x 10³/pL) and percent CD34+ was 0.09% to 0.36 % (mean: 0.22%) with absolute CD34+ counts of 20 to 80 pL (mean: 60 pL). Four of the mobilized subjects were successfully infused with SAR445136 at a single dose ranging from 3.2 to 9.7 x 10⁶ CD34+ HSPC/kg (mean: 5.17 x 10⁶ CD34+ HSPC/kg). All 4 subjects engrafted with a median time to platelet and neutrophil recovery of 24.5 and 21 .5 days, respectively. No rescue doses were required.

[0207] All 4 patients improved clinically since SAR445136 infusion. Measurements of HbF and HbS (mean and standard deviation) in all infused subjects are presented in FIG. 4. Total Hb stabilized at 9-10 g/dL by week 26 post-SAR445136 infusion along with improvements in the clinical markers of hemolysis in all 4 subjects.

[0208] The percent HbF levels were 1 % to 11% at screening, and increased to a range of 15% to 29% by Week 13 in all 4 subjects. The percent HbF levels increased to a range of 14% to 39% by Week 26 in all four subjects with at least 26 weeks follow up, and persisted at 35% in 1 subject at 65 weeks follow up (FIG. 4). The percent HbF level was 38% in one subject at 91 weeks.

[0209] Percent F cells increased to a range of 48% to 94% in all four subjects with at least 26 weeks follow up, and persisted at 99% in 1 subject with 91 weeks follow up. The fourth subject had 94% F cells at 26 weeks follow up. (FIG. 5).

[0210] All four treated subjects showed HbF/F cell levels above the threshold for preventing HbS polymerization. By week 26 all four treated subjects reached a level of greater than/equal to 10 pg of HbF/F cells, and this level was sustained in 2/3 patients with longer follow-up (FIG. 6). It has been recently suggested that there is a HbF/Fcell range above which with no HbS polymerization and no symptoms in HbS-HPFH (hereditary persistence of fetal hemoglobin) genotype are observed. (Steinberg MH. (2020) Blood. 136(21): 2392- 2400).

[0211] BCL11A Gene Modification: [0212] The SAR445136 investigational drug product had on-target BCL11A gene modification (61-78% indels) in all 4 subjects. The BCL11A gene modification results (% indels) for each of the 4 infused patients are shown in Tables 6-9. At 26 weeks post- SAR445136 infusion, the indel frequency ranged from 17-34% (mean =25%) in unsorted bone marrow in all 4 subjects. In the subject with 91 weeks follow up, the marrow indel frequency was 28% and 26% at weeks 26 and 52, respectively (see Table 6).

Table 6. BCL11A Gene Modification (% indels): Subject 103-002

Table 7. BCL11A Gene Modification (% indels): Subject 100-001

Table 8. BCL11A Gene Modification (% indels): Subject 102-001

Table 9. BCL11A Gene Modification (% indels): Subject 103-003

[0213] Safety and Tolerability:

[0214] Of the 4 subjects that have been infused, SAR445136 was generally well tolerated with no infusion related reactions. Reported AEs were consistent with plerixafor mobilization and busulfan myeloablation therapy. One SAE of sickle cell anemia with crisis (VOC) was reported about 9 months after SAR445136 infusion in 1 subject; no other SCD-related events were reported in the 4 subjects. (FIG. 7). No AEs or SAEs were reported as attributed to SAR445136.

[0215] Quality of Life:

[0216] These preliminary results showed a trend of improvement exceeding the proposed minimally clinically important difference in all PROMIS-57 HRQoL measured domains except for sleep disturbance and pain interference.

Conclusion

[0217] These preliminary data provide proof-of-concept (POC) efficacy and safety results and confirm the potential therapeutic value of ZFN-mediated modification of the BCL11A ESE region and SAR445136 infusion to address current unmet needs of patients with SCD. All 4 infused subjects showed increases in total Hb, HbF, and %F cells. SAR445136 was well tolerated in all four patients infused to date, with a single post-infusion VOC reported in one subject about 9 months after infusion. Clinical improvements in PROMIS-57 domains were also observed. There was natural enrichment of modified erythroid lineage cells in the blood with elevated HbF, sufficient for a clinical benefit. This suggests that modification of a minority of HSCs may be sufficient for a clinical benefit.

[0218] In conclusion, SAR445136 has the potential to induce hematologic changes in a subject and could provide a level of protection against the clinical sequelae of SCD.

[0219] All patents, patent applications and publications mentioned herein are hereby incorporated by reference in their entirety.

[0220] Although disclosure has been provided in some detail by way of illustration and example for the purposes of clarity of understanding, it will be apparent to those skilled in the art that various changes and modifications can be practiced without departing from the spirit or scope of the disclosure. Accordingly, the foregoing descriptions and examples should not be construed as limiting.

SUPPLEMENTAL TABLES

Table A: List of Prohibited Medications

Table B: Schedule of Events: Screening, Stem Cell Mobilization, Apheresis, and Conditioning

Abbreviations: AE=adverse event; DNA=deoxyribonucleic acid; DXA=dual-energy x-ray absorptiometry; ECG=electrocardiogram; ECHOechocardiogram; HbF=fetal hemoglobin; HbS=sickle hemoglobin; HSPC=hematopoietic stem and progenitor cell; ICF=informed consent form; IV=intravenous; MRA=magnetic resonance angiography; MRI=magnetic resonance imaging; MUGA= multi pie gated acquisition; PBL=peripheral blood lymphocyte; PBMC=peripheral blood mononuclear cell; PFT=pulmonary function test; PK= pharmacokinetics; PROMIS-57=Patient-Reported Outcomes Measurement Information System 57; SAE=serious adverse event; SCD=sickle cell disease; WBC=white blood cell

Footnotes:

1 Any scheduled vaccinations (e.g., influenza) were completed at least 30 days prior to the conditioning phase.

2 It was at the discretion of the Investigator, in consultation with the Medical Monitor, to waive any procedure if the procedure had been performed within the standard interval of scheduled study visits.

3 Reviewed select Inclusion/Exclusion criteria to ascertain subject's eligibility to continue in the study.

4 A minimum of 1 year of transfusion history prior to Screening was required to be eligible to participate in the study. Up to 3 years of history was recorded if available.

5 A physical examination was conducted on each subject at specified visits.

6 Conducted on Day 1 of stem cell mobilization.

7 Vital signs were monitored before, during, and after the infusion in accordance with study site practice.

8 For female subjects with childbearing potential only. Pregnancy testing was conducted on Day 1 of stem cell mobilization and within 2 days of conditioning.

9 Bone marrow aspiration was completed at the time of central IV catheter placement.

16 Subjects were monitored throughout the study for the potential development of hematopoietic malignancies using clinical history, physical examinations, laboratory assessments, and bone marrow aspirations.

17 Scheduling of gamete cryopreservation was initiated as soon as possible after mobilization. All procedures related to gamete cryopreservation were completed prior to busulfan administration.

18 Plerixafor dosing, timing, or route of administration may have been modified or discontinued at the discretion of the Investigator based on the clinical status of the subject or if the WBC exceeds 100,000/pL.

19 If the first apheresis cycle did not mobilize the minimum number of CD34+ HSPC required for both SAR445136 drug manufacturing and the rescue treatment, the stem cell mobilization procedure may have been repeated. The timing of repeat apheresis cycles were at the discretion of the Investigator based on the subject’s clinical status.

20 At the Baseline Visit, subjects may have had an optional busulfan PK assessment following a single low dose.

21 Subjects did not proceed to conditioning procedure with IV busulfan until a sufficient quantity of rescue treatment was obtained and SAR445136 was manufactured, passed quality control and release testing, and was confirmed as received at the study site.

23 Seizure prophylaxis was required starting prior to and continuing at least 24 hours after the final dose of IV busulfan conditioning. Iron- chelating agents were discontinued 2 weeks prior to busulfan administration and may be restarted after successful SAR445136 engraftment or at the discretion of the Investigator. Concomitant acetaminophen or metronidazole was avoided.

24 AE and SAE data was collected from signature of the ICF through the final study visit.

Table C: Schedule of Events: Post-Transplantation Study Period

Abbreviations: AE= adverse event; DXA=dual-energy x-ray absorptiometry; ECHOechocardiogram; EOS=end of study visit, HbF=fetal hemoglobin; HSPC=hematopoietic stem and progenitor cells; MUGA=multiple gated acquisition; PBL= peripheral blood lymphocytes; PBMC=peripheral blood mononuclear cells; PFT=pulmonary function test; PROMIS-57=patient-reported outcomes measurement information system 57; SAE= serious adverse event; SCD=sickle cell disease; Wk=week

Footnotes:

1 A physical examination was conducted on each subject at specified visits.

2 Unscheduled bone marrow aspiration may also be conducted at any time if clinically indicated, e.g., to evaluate the potential development of hematological malignancy.

3 Dose of SAR445136 was administered IV as a 1-time dose no sooner than 72 hours after the final dose of IV busulfan in the conditioning phase.

4 More frequent monitoring if the prevalence of a single allele is >10% was at the discretion of the Investigator.

5 Vital signs were monitored before, during, and after the infusion and in accordance with study site practice.

6 AE and SAE data were collected from mobilization through final study visit.

Claims

CLAIMS What is claimed is:

1. An ex vivo method of treating sickle cell disease (SCD) in a subject, the method comprising: administering to the subject in need thereof a composition comprising a genetically modified cell comprising a red blood cell (RBC) precursor cell, wherein the precursor cell comprises SB-mRENH1 mRNAs and SB-mRENH2 mRNAs, which encode a ZFN pair; and a genomic modification made following cleavage by the ZFN pair, wherein the modification is within an endogenous BCL11A enhancer sequence.

2. The method of claim 1, wherein the sickle cell disease is severe sickle cell disease.

3. The method of claim 1 or 2, wherein the subject has adequate physiological function by BMT CNT guidelines prior to administering of the composition.

4. The method of any one of claims 1-3, wherein the composition further comprises a cell descended from the genetically modified cell comprising the SB-mRENH1 mRNAs and the SB-mRENH2 mRNAs.

5. The method of any one of claims 1-4, wherein the fetal hemoglobin (HbF) production in the subject is increased after administration of the composition.

6. The method of any one of claims 1-5, wherein one or more clinical symptoms of sickle cell disease in the subject are decreased, ameliorated, or eliminated.

7. The method of claim 6, wherein one of the clinical symptoms in the subject that is decreased, ameliorated, or eliminated is vaso-occlusive crisis (VOC).

8. The method of any one of claims 1-7, wherein a change from baseline of clinical laboratory hemoglobin fractions in grams/dL plasma and/or percent HbF of total hemoglobin (Hb) is achieved in the subject.

9. The method of any one of claims 1-8, wherein levels of SCD-related disease biomarkers in the subject are altered following treatment.

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10. The method of claim 9, wherein the biomarkers are changes in iron metabolism and/or changes in levels of erythropoietin, haptoglobin and/or hepcidin.

11. The method of any one of claims 1-10, wherein the clinical symptoms associated with iron overload or associated with baseline transfusion therapy in the subject are ameliorated or eliminated.

12. The method of any one of claims 1-11, wherein the need for RBC transfusions and infusion platelet transfusion, intravenous immunoglobin (I VIG) transfusion, plasma transfusion and/or granulocyte transfusion in the subject is reduced or eliminated.

13. The method of any one of claims 1-12, wherein the number and/or percent of F cells in the subject is modified following administration of the composition.

14. The method of any one of claims 1-13, wherein the genetically modified cells are autologous or allogeneic.

15. The method of any one of claims 1-14, wherein the BCL11A-genetically modified cells further comprise one or more additional genetic modifications.

16. The method of any one of claims 1-13, wherein the genetically modified cells are hematopoietic stem cells isolated from the subject.

17. The method of claim 16, wherein the hematopoietic stem cells are CD34+ hematopoietic stem or precursor cells (HSC/PC) and the CD34+ HSC/PC are mobilized in the subject by treatment with one or more doses of plerixafor.

18. The method of claim 17, wherein at least 25 x 10⁶ CD34+ HSPCs/kg are mobilized in the subject and the mobilized cells are harvested by one or more apheresis cycles.

19. The method of any one of claims 1-18, further comprising administering one or more myeloablative condition agents to the subject prior to administration of the composition comprising the genetically modified cells.

20. The method of claim 19, wherein the myeloablative agent comprises busulfan.

21. The method of any one of claims 1-20, wherein the dose of genetically modified cells administered to the subject is between about 3 x 10⁶ cells/kg and 20 x 10⁶ cells/kg.

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22. The method of any one of claims 1-20, wherein the composition administered to the subject comprises a dose of cells between about 3.2 x 10⁶ cells/kg and about 9.7 x 10⁶ cells/kg.

23. The method of any one of claims 1-20, wherein the composition administered to the subject comprises a dose of cells about 5.17 x 10⁶ cells/kg.

24. The method of any one of claims 1-23, wherein the genetically modified cells administered to the subject are formulated with approximately 1.0-2.0 x 10⁸ cells per bag at a concentration of approximately 1 x 10⁷ cells/mL.

25. The method of any one of claims 1-24, wherein the genetically modified cells are cryopreserved prior to administration to the subject.

26. The method of any one of claims 1-25, further comprising monitoring the subject’s vital signs prior to, during and/or after administration of the genetically modified cells.

27. The method of any one of claims 1-26, further comprising assessing hemoglobin, neutrophil and/or platelet levels in the subject prior to administration of the genetically modified cells to determine baseline levels of hemoglobin in the subject.

28. The method of claim 27, wherein hemoglobin, neutrophil and/or platelet levels in the subject after administration of the genetically modified cells increase or remain stable as compared to baseline levels for weeks or months after administration.

29. The method of any one of claims 1-28, further comprising assessing the subject using a health-related quality of life (HRQoL) survey.

30. The method of any one of claims 1-29, further comprising assessing the subject using the Patient-Reported Outcomes Measurement Information System 57 (PROMIS-57).

31 . The method of any one of claims 1-30, further comprising assessing the subject using the Karnofsky Performance Scale.

32. The method of any one of claims 1-31 , wherein the subject receives one or more packed red blood cell (PRBC) transfusions prior to and/or after administration of the genetically modified cells.

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33. The method of any one of claims 1-32, wherein the need for additional therapies such as a bone marrow transplant, blood component and/or iron chelation therapy PRBC transfusions in the subject is reduced or eliminated.

34. The method of claim 33, wherein the need for additional therapies is reduced or eliminated within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more days of administration of the genetically modified cells.

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