B cells genetically engineered to secrete follistatin and methods of using the same to treat follistatin-related diseases, conditions, disorders and to enhance muscle growth and strength

Abstract

To provide B cells genetically engineered to secrete follistatin and methods of using the same to treat follistatin-related diseases, conditions, and disorders and to enhance muscle growth and strength.SOLUTION: The present invention relates to methods for administering autologous and / or allogeneic B cells that have been genetically modified to produce a therapeutic agent, such as follistatin. Specifically disclosed are methods for administering a single maximally effective dose of genetically modified B cells, and methods for administering multiple doses of genetically modified B cells expressing follistatin. The compositions and methods disclosed herein are useful for long-term invivo delivery of follistatin.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 644,362, filed March 16, 2018, and U.S. Provisional Application No. 62 / 644,356, filed March 16, 2018, each of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Statement The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The text file containing the sequence listing is named IMCO-008_01WO_ST25.txt. This text file is 12 KB, was created on March 18, 2019, and was submitted electronically via EFS-Web.

[0003] background Technical Field The present disclosure relates to the use of B cells for long-term in vivo delivery of therapeutic agents, such as follistatin, and in particular to the administration of single and multiple doses of B cells to a subject (e.g., a human). [Background technology]

[0004] 2. Description of Related Art Muscular dystrophy (MD) is a progressive, inherited neuromuscular disorder characterized by muscle wasting and weakness (Emery (2002) The Lancet, 359:687-695). Many forms of muscular dystrophy are fatal and currently untreatable.

[0005] Duchenne muscular dystrophy (DMD) is the most common X-linked neuromuscular disorder. The disease is caused by mutations in the DMD gene, which encodes dystrophin. The alteration or absence of this protein results in abnormal sarcolemmal ruptures. The disease is characterized by abnormal variations in the diameter of muscle fibers (atrophic and hypertrophic fibers) in proximal muscles and progressive muscle damage. Damaged muscle releases the intracellular enzyme creatine kinase (CK). As a result, DMD patients are characterized by elevated serum CK levels (up to 10-fold higher than normal). The pathophysiological cascade consists of tissue inflammation, myofiber necrosis, and replacement of muscle with fibro-fatty tissue.

[0006] Alternative allelic variants of the DMD gene cause a milder form of MD known as Becker muscular dystrophy (BMD), which is clinically similar to DMD, but the onset of symptoms occurs later in life.

[0007] Many medications have been tried in MD, but none have proven effective in arresting the course of the disease. Current treatment modalities remain within the realm of physical therapy and rehabilitation.

[0008] Several clinical trials using corticosteroids (e.g., prednisone and / or its derivatives) have demonstrated improvement in individuals with MD, particularly in the short term. Although the exact mechanism by which corticosteroids alleviate the disease phenotype is unclear, corticosteroids are thought to act by reducing inflammation, suppressing the immune system, improving calcium homeostasis, upregulating the expression of compensatory proteins, and increasing myoblast proliferation (Khurana et al. (2003) Nat. Rev. Drug Discovery 2:279-386). However, corticosteroids administered over time can induce muscle atrophy, which may primarily affect proximal muscles, the very same muscles affected in DMD and BMD. Corticosteroid-induced muscle effects and other side effects may limit the long-term effectiveness of corticosteroid therapy.

[0009] The transforming growth factor-beta (TGF-beta) superfamily contains a variety of growth factors that share common sequence elements and structural motifs. These proteins are known to exert biological effects on diverse cell types in both vertebrates and invertebrates. Members of this superfamily play important functions during embryonic development in pattern formation and tissue specification and can influence various differentiation processes, including adipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis, and epithelial cell differentiation. This family is divided into two general branches: the BMP / GDF and the TGF-beta / activin / BMP10 branches, and these members have diverse, often complementary, actions. Manipulating the activity of TGF-beta family members can often result in significant physiological changes in the organism. For example, Piedmontese and Belgian Blue cattle breeds carry loss-of-function mutations in the GDF8 (also known as myostatin) gene, which causes a significant increase in muscle mass. Grobet et al., Nat. Genet. 1997, 17(1):71-4. Furthermore, in humans Inactive alleles of GDF8 are associated with increased muscle mass and, reportedly, exceptional strength. Schuelke et al., N Engl J Med 2004, 350:2682-8. Furthermore, mice genetically engineered to express dominant-negative activin receptor IIB (ActRIIB) or to express follistatin have exceptional muscle mass (Lee, SJ and McPherron, AC, Proc Natl Acad Sci U S A. 2001 Jul 31;98(16):9306-11), and overexpression of follistatin in non-human primates enhances muscle growth and strength. is enhanced. Kota J, et al., Sci Transl Med. 2009 Nov 11;1(6).

[0010] Thus, there is a need for methods of delivering agents that function as effective modulators of TGF-beta signaling. Current methods for treating chronic diseases and disorders include direct injection of therapeutic agents (e.g., therapeutic polypeptides), viral vector-mediated gene therapy, and adoptive transfer of stem cells (e.g., hematopoietic stem cell transfer). However, each of these methods has disadvantages. Injection of recombinant therapeutic proteins suffers from the finite half-life of the protein, and all three methods result in suboptimal tissue penetration by the therapeutic agent. Altering endogenous tissues to produce therapeutic agents, such as by injection of recombinant adeno-associated virus (AAV) and lentiviral vectors, generally results in the therapeutic agent being produced from a centralized location. Production of the therapeutic agent from a single location increases the probability of localized toxicity in the producing tissue. Furthermore, because recombinant viruses are considered foreign, it is unlikely that viral vectors can be administered multiple times without causing adverse reactions, meaning there is a single injection opportunity to achieve a precise dose of the therapeutic agent. Given the biological variability inherent in procedures such as the in vivo transfer of nucleic acids into cells using viruses, achieving a desired dose within the constraints of a single injection would be highly unreliable. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Emery (2002) The Lancet, 359:687-695 [Non-patent document 2] Khurana et al. (2003) Nat. Rev. Drug Discovery 2:279-386 [Non-patent document 3] Grobet et al., Nat. Genet. 1997, 17(1):71-4 [Non-patent document 4] Schuelke et al., N Engl J Med 2004, 350:2682-8 [Non-Patent Document 5] Lee, SJ and McPherron, AC, Proc Natl Acad Sci US A. 2001 Jul 31;98(16):9306-11 [Non-patent document 6] Kota J, et al., Sci Transl Med. 2009 Nov 11;1(6) Summary of the Invention [Problem to be solved by the invention]

[0012] Thus, there remains a need in the art for long-term treatments for the many chronic diseases and disorders associated with TGF-beta signaling. [Means for solving the problem]

[0013] Overview of the embodiment The present disclosure generally relates to compositions and methods for administering and dosing genetically modified B cell compositions to treat chronic diseases and disorders. In various embodiments, the present disclosure provides compositions and methods for administering and dosing B cells genetically modified to express a polypeptide (e.g., a follistatin polypeptide) capable of modulating TGF-beta signaling. In certain embodiments, the present disclosure relates to compositions and methods for administering and dosing B cells genetically modified to express a follistatin polypeptide. In certain embodiments, the present disclosure relates to compositions and methods for administering and dosing B cells genetically modified to express a follistatin polypeptide having the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. Such B cells can be used in various embodiments, for example, to increase muscle size or strength in a subject (e.g., a human). The present disclosure provides these and other advantages, as described in the detailed description.

[0014] In some embodiments, the present invention provides recombinant B cells comprising a follistatin gene. In some embodiments, the follistatin gene is operably linked to a promoter. In some embodiments, the follistatin gene is a human follistatin gene. In some embodiments, the follistatin gene is a splice site variant of human follistatin FST-344. In some embodiments, the B cells are human B cells. In some embodiments, the B cells are transduced with the follistatin gene. In some embodiments, the B cells contain the follistatin gene because they have been transduced with the follistatin gene using the Sleeping Beauty transposon system. In some embodiments, the B cells express the follistatin gene by transduction with a virus carrying the follistatin gene. In some embodiments, the B cells contain the follistatin gene because they have been transduced with a retrovirus, lentivirus, adenovirus, or adeno-associated virus containing the follistatin gene. In some embodiments, B cells are engineered to contain the follistatin gene using targeted integration techniques. In some embodiments, targeted integration utilizes one or more zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and / or CRISPR / Cas systems, including but not limited to CRISPR / Cas9 systems. In some embodiments, B cells are engineered to contain the follistatin gene by introducing a nucleic acid encoding follistatin using a method selected from the group consisting of a retroviral vector, a lentiviral vector, an adeno-associated viral vector, an adenoviral vector, or any other RNA or DNA viral vector. In some embodiments, B cells are engineered to contain the follistatin gene using chemical or physical means such as lipofection, polycation complex formation, electroporation, etc. In some embodiments, the recombinant B cells are engineered to contain non-viral DNA and / or RNA encoding the follistatin gene. In some embodiments, the follistatin gene is secreted by the recombinant B cells. In some embodiments, the recombinant B cells are derived from B cells obtained from the subject or B cells derived from cells obtained from the subject. In some embodiments, the recombinant B cells are derived from precursor B cells obtained from the subject. In some embodiments, the recombinant B cells are derived from cells obtained from the subject that have differentiated into B cells or precursor B cells. In some embodiments, the recombinant B cells are (a) collecting and isolating immune cells from the blood of a subject; (b) transducing the cells with DNA encoding follistatin; (c) expanding the selected cells ex vivo; (d) Differentiating the ex vivo expanded cells into plasma cells and / or plasmablasts. and is operated by.

[0015] In some embodiments, the immune cells isolated from step a are CD19-positive cells. In some embodiments, the transduction step of step b is by electroporation. In some embodiments, the electroporation utilized the Sleeping Beauty transposon system. In some embodiments, the differentiated cells are CD38(+) and CD20(-). In some embodiments, the invention provides methods comprising administering such recombinant B cells to a subject.

[0016] In some embodiments, the present invention provides methods of delivering follistatin to a subject in need thereof, the method comprising administering recombinant B cells comprising a follistatin gene. In some embodiments, the present invention provides methods of delivering follistatin to a subject in need thereof, the method comprising administering to the subject any one of the recombinant B cells disclosed herein that express a follistatin polypeptide. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject has a muscular disorder. In some embodiments, the muscular disorder is a muscular dystrophy. In some embodiments, the muscular dystrophy is selected from Duchenne muscular dystrophy, Becker muscular dystrophy, and fascioscapulohumeral muscular dystrophy. In some embodiments, the muscle disorder is an inflammatory myopathy. In some embodiments, the inflammatory myopathy is inclusion body myositis. In some embodiments, the muscle disorder is muscle injury or muscle trauma. In some embodiments, the muscle disorder is muscle disuse. In some embodiments, the muscle disuse occurs after prolonged bed rest or limb immobilization. In some embodiments, the muscle disorder is muscle atrophy or muscle weakness. In some embodiments, the muscle atrophy or muscle weakness is caused by aging, cancer, or chronic disease. In some embodiments, the muscle atrophy or muscle weakness is caused by sarcopenia. In some embodiments, the muscle atrophy or muscle weakness is caused by spinal muscular atrophy (SMA). In some embodiments, the muscle atrophy or muscle weakness is caused by amyotrophic lateral sclerosis (ALS). In some embodiments, the muscle atrophy or muscle weakness is caused by Pompe disease. In some embodiments, the subject has healthy muscles. In some embodiments, administration of B cells that express a follistatin polypeptide to a subject having healthy muscles increases the size or strength of the subject's muscles.

[0017] In certain embodiments, the present disclosure provides a method for treating muscular dystrophy, the method comprising administering B cells expressing a follistatin polypeptide to a subject with muscular dystrophy. In some embodiments, the subject has Becker muscular dystrophy. In some embodiments, administering the recombinant B cells to the subject results in treatment of a disease, disorder, or condition in the subject. In some embodiments, administering the recombinant B cells to the subject results in treatment of muscular dystrophy. In some embodiments, administering the recombinant B cells to the subject results in the subject gaining weight. In some embodiments, the subject gains weight by at least about 4%. In some embodiments, a significant weight gain occurs within 30 days. In some embodiments, a significant weight gain occurs over about 30 days. In some embodiments, administering the recombinant B cells to the subject increases the muscle mass of the subject. In some embodiments, administering the recombinant B cells to the subject increases the strength of the subject. In some embodiments, administering the recombinant B cells to the subject increases the plasma level of follistatin in the subject.

[0018] In some embodiments, the present invention provides methods of treating, preventing, or alleviating muscular dystrophy by administering recombinant B cells comprising a follistatin gene. In some embodiments, the method of treating, preventing, or alleviating muscular dystrophy comprises administering any one of the recombinant B cells disclosed herein. In some embodiments, the method comprises administering two or more sequential doses of genetically modified B cells to a subject. In some embodiments, the administering step comprises two or more doses of genetically modified B cells at a suboptimal single dose concentration. In some embodiments, the administering step comprises three or more doses of genetically modified B cells. In some embodiments, the genetically modified B cells are autologous to the subject. In some embodiments, the genetically modified B cells are allogeneic to the subject. In some embodiments, the subject is human. In some embodiments, the genetically modified B cells are CD20-, CD38-, and CD138-. In some embodiments, the genetically modified B cells are CD20-, CD38+, and CD138+. In some embodiments, the genetically modified B cells are CD20-, CD38+, and CD138-. In some embodiments, the administering step comprises intravenous, intraperitoneal, subcutaneous, or intramuscular injection. In some embodiments, the administering step comprises intravenous injection. In some embodiments, the genetically modified B cells are engineered on day 2 or day 3 of culture. In some embodiments, the genetically modified B cells are engineered using a method comprising electroporation. In some embodiments, the genetically modified B cells are collected for administration to a subject on day 4, day 5, day 6, or day 7 of culture after manipulation. In some embodiments, the genetically modified B cells are collected for administration to a subject on day 8 or more of culture after manipulation. In some embodiments, the genetically modified B cells are collected for administration to a subject on day 10 or more of culture after manipulation. In some embodiments, the collected genetically modified B cells do not produce significant levels of inflammatory cytokines. In some embodiments, the genetically modified B cells are collected at a time during culture at which they are determined not to produce significant levels of inflammatory cytokines.In some embodiments, the genetically modified B cells are grown in a culture system containing each of IL-2, IL-4, IL-10, IL-15, IL-31, and multimerized CD40 ligand throughout the pre- and post-manipulation culture periods. In some embodiments, the multimerized CD40 ligand is a HIS-tagged CD40 ligand that is multimerized using an anti-His antibody. In some embodiments, the method further comprises expanding the genetically modified B cells prior to administering to the subject. In some embodiments, the final population of expanded genetically modified B cells exhibits a high degree of polyclonality. In some embodiments, any particular B cell clone in the final population of expanded genetically modified B cells comprises less than 0.2% of the total B cell population. In some embodiments, any particular B cell clone in the final population of expanded genetically modified B cells comprises less than 0.05% of the total B cell population. In some embodiments, the genetically modified B cells comprise a polynucleotide encoding a human DHFR gene that has enhanced resistance to methotrexate. In some embodiments, the human DHFR gene with enhanced resistance to methotrexate contains a leucine to tyrosine substitution mutation at amino acid 22 and a phenylalanine to serine substitution mutation at amino acid 31. In some embodiments, the method includes treating the genetically modified B cells with methotrexate prior to collection for administration. In some embodiments, the methotrexate treatment is between 100 nM and 300 nM. In some embodiments, the methotrexate treatment is 200 nM. In some embodiments, the genetically modified B cells migrate to various tissues upon administration to a subject. In some embodiments of the method, at least one genetically modified B cell of the population of genetically modified B cells administered to the subject migrates to one or more tissues selected from the group consisting of bone marrow, intestine, muscle, spleen, kidney, heart, liver, lung, and brain. In some embodiments of the method, at least one genetically modified B cell of the population of genetically modified B cells administered to the subject migrates to the bone marrow, intestine, muscle, spleen, kidney, heart, liver, lung, and brain of the subject.

[0019] In some embodiments, the present invention provides modified B cells transduced to express both the follistatin gene and the dihydrofolate reductase (DHFR) gene. In certain embodiments, for example, the following are provided: (Item 1) Recombinant B cells containing the follistatin gene. (Item 2) 2. The B cell of item 1, wherein the follistatin gene is operably linked to a promoter. (Item 3) 3. The B cell of item 1 or 2, wherein the follistatin gene is a human follistatin gene. (Item 4) 4. The B cell of any one of items 1 to 3, wherein the follistatin gene is a splice site variant of human follistatin FST-344. (Item 5) 8. The B cell of any one of the preceding items, wherein the B cell is a human B cell. (Item 6) 8. The B cell of any one of the preceding items, wherein the B cell is transduced or rearranged with the follistatin gene. (Item 7) 7. The B cell of any one of items 1 to 6, wherein the B cell comprises the follistatin gene because the B cell has been transduced with the follistatin gene using a transposon system. (Item 8) 8. The B cell of item 7, wherein the transposon system is a Sleeping Beauty transposon system or a Piggybac transposon system. (Item 9) 8. The B cell of any one of items 1 to 7, wherein the B cell expresses the follistatin gene by transduction with a virus carrying the follistatin gene. (Item 10) 8. The B cell of any one of items 1 to 7, wherein the B cell comprises the follistatin gene because the B cell has been transduced with a retrovirus, lentivirus, adenovirus, or adeno-associated virus comprising the follistatin gene. (Item 11) 8. The B cell of any one of items 1 to 7, wherein the B cell is engineered to contain the follistatin gene using a targeted integration approach. (Item 12) 12. The B cell of item 11, wherein the targeted integration utilizes one or more zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and / or a CRISPR / Cas system, including, but not limited to, a CRISPR / Cas9 system. (Item 13) 8. The B cell of any one of items 1 to 7, wherein the B cell is engineered to contain non-viral DNA and / or RNA encoding follistatin introduced using chemical or physical means such as lipofection, polycation complexation, electroporation, etc. by introducing a nucleic acid encoding follistatin using a method selected from the group consisting of a retroviral vector, a lentiviral vector, an adeno-associated viral vector, an adenoviral vector, or any other RNA or DNA viral vector. (Item 14) 10. The B cell of any one of the preceding items, wherein the follistatin protein is secreted by the recombinant B cell. (Item 15) 1. A method of delivering follistatin to a subject, comprising administering recombinant B cells comprising a follistatin gene. (Item 16) 15. A method of delivering follistatin to a subject in need thereof, comprising administering the recombinant B cell of any one of items 1 to 14. (Item 17) 17. The method of claim 15 or 16, wherein the subject is a mammal. (Item 18) 18. The method of any one of items 15 to 17, wherein the subject is a human. (Item 19) 19. The method of any one of items 15 to 18, wherein the subject has muscular dystrophy. (Item 20) 20. The method of any one of items 15 to 19, wherein the subject has Becker muscular dystrophy. (Item 21) 21. The method of any one of items 15 to 20, wherein the administering of the recombinant B cells to the subject results in treatment of a disease, disorder, or condition in the subject. (Item 22) 21. The method of any one of items 15 to 20, wherein said administering said recombinant B cells to said subject results in treatment of muscular dystrophy. (Item 23) 23. The method of any one of items 15 to 22, wherein said administering said recombinant B cells to said subject causes said subject to gain weight. (Item 24) 24. The method of claim 23, wherein the subject gains at least about 4% body weight. (Item 25) 25. The method of item 24, wherein significant weight gain occurs within 30 days. (Item 26) 25. The method of claim 24, wherein the significant weight gain occurs over a period of about 30 days. (Item 27) 27. The method of any one of items 15 to 26, wherein said administering said recombinant B cells to said subject increases muscle mass in said subject. (Item 28) 28. The method of any one of items 15 to 27, wherein said administering said recombinant B cells to said subject results in greater strength in said subject. (Item 29) 29. The method of any one of items 15 to 28, wherein said administering of said recombinant B cells increases follistatin plasma levels in said subject. (Item 30) A method of treating, preventing, or alleviating muscle disorders by administering recombinant B cells containing the follistatin gene. (Item 31) 14. A method of treating, preventing, or alleviating muscular dystrophy by administering the recombinant B cell of any one of items 1 to 13. (Item 32) 33. The recombinant B cell of any one of claims 1 to 13, wherein the recombinant B cell is derived from a B cell obtained from the subject or a B cell derived from a cell obtained from the subject. 33. The recombinant B cell of item 32, wherein the recombinant B cell is derived from a precursor B cell obtained from the subject. (Item 34) 33. The recombinant B cell of claim 32, wherein the recombinant B cell is derived from a cell obtained from the subject that has differentiated into the B cell or a precursor cell of a B cell. (Item 35) the recombinant B cells (a) collecting and isolating immune cells from the blood of the subject; (b) transducing the cells with DNA encoding the follistatin; (c) expanding the selected cells ex vivo; (d) differentiating the expanded cells ex vivo into plasma cells and / or plasmablasts. 35. The recombinant B cell of any one of items 1 to 13 and 32 to 34, wherein the recombinant B cell is engineered by: (Item 36) 36. The recombinant B cell of item 35, wherein the immune cells isolated from step a are CD19-positive cells. (Item 37) 37. The recombinant B cell according to item 35 or 36, wherein the transducing step in step b is by electroporation. (Item 38) 38. The recombinant B cell of item 37, wherein the electroporation utilizes the Sleeping Beauty transposon system. (Item 39) 39. The recombinant B cell of any one of items 35 to 38, wherein the differentiated cells are CD38(+) and CD20(-). (Item 40) 40. A method comprising administering to a subject the recombinant B cell of any one of items 35 to 39. (Item 41) 41. The method of any one of paragraphs 15 to 31 and 35 to 40, comprising administering to the subject two or more sequential doses of the genetically modified B cells. (Item 42) 42. The method of claim 41, wherein the administering step comprises two or more doses of the genetically modified B cells at a suboptimal single dose concentration. (Item 43) 42. The method of claim 41, wherein the administering step comprises three or more doses of genetically modified B cells. (Item 44) 42. The method of claim 41, wherein the genetically modified B cells are autologous to the subject. (Item 45) 42. The method of claim 41, wherein the genetically modified B cells are allogeneic to the subject. (Item 46) 42. The method of claim 41, wherein the subject is a human. (Item 47) 42. The method of claim 41, wherein the genetically modified B cells are CD20-, CD38- and CD138-. (Item 48) 42. The method of claim 41, wherein the genetically modified B cells are CD20-, CD38+, and CD138+. (Item 49) 42. The method of claim 41, wherein the genetically modified B cells are CD20-, CD38+ and CD138-. (Item 50) 42. The method of claim 41, wherein the administering step comprises intravenous, intraperitoneal, subcutaneous, intrathecal, intracameral, or intramuscular injection. (Item 51) 51. The method of claim 50, wherein the administering step comprises intravenous injection. (Item 52) 52. The method of any one of paragraphs 15 to 31 and 35 to 51, wherein the genetically modified B cells are manipulated on day 2 or day 3 after culture. (Item 53) 53. The method of claim 52, wherein the genetically modified B cells are engineered using a method comprising electroporation. (Item 54) (a) the genetically modified B cells are collected for administration to the subject on a day ranging from day 1 to day 12 of in vitro culture; (b) the genetically modified B cells are harvested for administration to the subject on day 4, day 5, day 6, day 7, or day 8 of culture after manipulation; 54. The method according to any one of items 15 to 31 and 35 to 53. (Item 55) 55. The method of any one of paragraphs 15 to 31 and 35 to 54, wherein the genetically modified B cells are harvested for administration to the subject on or after day 8 from the initiation of culture after manipulation. (Item 56) 56. The method of claim 55, wherein the genetically modified B cells are harvested for administration to the subject on or before day 10 from the initiation of culture after manipulation. (Item 57) 57. The method of any one of paragraphs 15 to 31 and 35 to 56, wherein the collected genetically modified B cells do not produce significant levels of inflammatory cytokines. (Item 58) 58. The method of any one of paragraphs 15 to 31 and 35 to 57, wherein the genetically modified B cells are harvested at a time during culture when they are determined not to produce significant levels of inflammatory cytokines. (Item 59) 59. The method of any one of paragraphs 15 to 31 and 35 to 58, wherein the genetically modified B cells are grown in a culture system comprising each of IL-2, IL-4, IL-10, IL-15, IL-31 and multimerized CD40 ligand throughout the pre- and post-manipulation culture periods. (Item 60) 60. The method of claim 59, wherein the multimerized CD40 ligand is a HIS-tagged CD40 ligand that is multimerized using an anti-his antibody. (Item 61) 61. The method of any one of paragraphs 15 to 31 and 35 to 60, further comprising expanding the genetically modified B cells prior to administering to the subject. (Item 62) 62. The method of claim 61, wherein the final population of expanded genetically modified B cells exhibits a high degree of polyclonality. (Item 63) 62. The method of paragraph 61, wherein any particular B cell clone in the final population of expanded genetically modified B cells comprises less than 0.2% of the total B cell population. (Item 64) 62. The method of paragraph 61, wherein any particular B cell clone in the final population of expanded genetically modified B cells comprises less than 0.05% of the total B cell population. (Item 65) 65. The method of any one of paragraphs 15 to 31 and 35 to 64, wherein the genetically modified B cells comprise a polynucleotide encoding a selectable marker. (Item 66) 66. The method of claim 65, wherein the selectable marker is a human DHFR gene that confers enhanced resistance to methotrexate. (Item 67) 67. The method of item 66, wherein the human DHFR gene having enhanced resistance to methotrexate contains a leucine to tyrosine substitution mutation at amino acid 22 and a phenylalanine to serine substitution mutation at amino acid 31. (Item 68) 68. The method of any one of paragraphs 15 to 31 and 35 to 67, comprising treating the genetically modified B cells with methotrexate prior to collection for administration. (Item 69) Item 69. The method of item 68, wherein the methotrexate treatment is between 100 nM and 300 nM. (Item 70) 70. The method of claim 69, wherein the methotrexate treatment is 200 nM. (Item 71) 71. The method of any one of paragraphs 15 to 31 and 35 to 70, wherein the genetically modified B cells migrate to a variety of tissues upon administration to the subject. (Item 72) 72. The method of any one of paragraphs 15 to 31 and 35 to 71, wherein at least one genetically modified B cell of the population of genetically modified B cells administered to the subject migrates to one or more tissues selected from the group consisting of bone marrow, intestine, muscle, spleen, kidney, heart, liver, lung, and brain. (Item 73) 73. The method of claim 72, wherein at least one genetically modified B cell of the population of genetically modified B cells administered to the subject migrates to the bone marrow, intestine, muscle, spleen, kidney, heart, liver, lung, and brain of the subject. (Item 74) Modified B cells transduced to express the follistatin gene and the DHFR gene. (Item 75) 1. A method for treating a muscle disorder, comprising administering to a subject B cells genetically modified to express follistatin. (Item 76) 76. The method of claim 75, wherein the muscle disorder is selected from muscular dystrophy, inflammatory myopathy, muscle injury or trauma, muscle disuse, and muscle atrophy or weakness. (Item 77) Item 78. The method according to Item 75 or 76, wherein the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, or facioscapulohumeral muscular dystrophy. 77. The method of item 75 or 76, wherein the inflammatory myopathic disorder is inclusion body myositis. (Item 79) 77. The method of item 75 or 76, wherein the muscle disuse occurs after prolonged bed rest or limb immobilization. (Item 80) 77. The method of item 75 or 76, wherein the muscle atrophy or weakness is caused by aging, cancer or a chronic disease. (Item 81) 76. The method of claim 75, wherein the muscle disorder is sarcopenia. (Item 82) 76. The method of claim 75, wherein the muscle disorder is spinal muscular atrophy (SMA). (Item 83) 76. The method of claim 75, wherein the muscle disorder is amyotrophic lateral sclerosis (ALS). (Item 84) 76. The method of item 75, wherein the muscle disorder is Pompe disease. (Item 85) 85. The method of any one of items 75 to 84, wherein the follistatin comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4. [Brief explanation of the drawings]

[0020] [Figure 1] Figure 1 shows that treatment with follistatin-expressing B cells results in increased follistatin levels in mouse plasma. From left to right in each of the control and treatment groups: bars correspond to days 21, 28, and 35, respectively.

[0021] [Figure 2A-B] Figure 2 shows that follistatin plasma levels in mice treated with follistatin-expressing B cells correlate with levels of human IgG, a surrogate marker for engraftment. Figures 2A-2D show follistatin plasma levels in four separate mice treated with follistatin-expressing B cells. [Figure 2C-D]Figure 2 shows that follistatin plasma levels in mice treated with follistatin-expressing B cells correlate with levels of human IgG, a surrogate marker for engraftment. Figures 2A-2D show follistatin plasma levels in four separate mice treated with follistatin-expressing B cells.

[0022] [Figure 3] FIG. 3 shows the percent change in body weight of mice treated or not with follistatin-expressing B cells.

[0023] [Figure 4A] Figure 4 shows strength assessments of mice treated or not with follistatin-expressing B cells. Figure 4A shows strength assessed by the forelimb grip test. Figure 4B shows strength assessed by the four-limb grip test. Figure 4C shows strength assessed by the hanging test. The percent improvement given below the graphs indicates the average percent improvement in the treated group compared to the untreated group. [Figure 4B-C] Figure 4 shows strength assessments of mice treated or not with follistatin-expressing B cells. Figure 4A shows strength assessed by the forelimb grip test. Figure 4B shows strength assessed by the four-limb grip test. Figure 4C shows strength assessed by the hanging test. The percent improvement given below the graphs indicates the average percent improvement in the treated group compared to the untreated group.

[0024] [Figure 5] Figure 5 shows in vitro follistatin expression in follistatin-expressing B cells. Figure 5A shows follistatin protein expression as determined by ELISA. Figure 5B shows follistatin mRNA expression as determined by RT-PCR. DETAILED DESCRIPTION OF THE INVENTION

[0025] Detailed Description The practice of the present invention will employ, unless specifically indicated to the contrary, conventional methods of molecular biology, recombinant DNA techniques, protein expression and protein / peptide / carbohydrate chemistry within the skill of those in the art, many of which are described below for illustrative purposes. Such techniques are fully explained in the literature. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., 2000); DNA Cloning: A Practical Approach, Vols. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Oligonucleotide Synthesis: Methods and Applications (P. Herdewijn, ed., 2004); Nucleic Acid Hybridization (B. Hames and S. Higgins, eds., 1985); Nucleic Acid Hybridization: Modern Applications (Buzdin and Lukyanov, eds., 2009); Transcription and Translation (B. Hames and S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Freshney, RI (2005) Culture of Animal Cells, a Manual of Basic Technique, 5th Edition, Hoboken NJ, John Wiley & Sons;B. Perbal, A Practical Guide to Molecular See, for example, "Cloning" (3rd ed., 2010); Farrell, R., RNA Methodologies: A Laboratory Guide for Isolation and Characterization (3rd ed., 2005). The publications discussed above are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0026] Definitions and Abbreviations Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used in this specification and the appended claims, unless specified to the contrary, the following terms have the meanings indicated. With respect to this specification, whenever a definition of a term defined herein differs from the definition given for this same term in an incorporated reference, the definition explicitly defined herein is the precise definition of the term.

[0027] The words "a" and "an" denote one or more unless otherwise noted.

[0028] "About" means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In any embodiment described in the context of a numerical value used in conjunction with the term "about," it is specifically contemplated that the term about may be omitted.

[0029] A "composition" can include an active agent and a carrier, inert or active, e.g., a pharmaceutically acceptable carrier, diluent, or excipient. In certain embodiments, the composition is sterile, substantially free of endotoxin, or non-toxic to a recipient at the dosage or concentration employed.

[0030] Unless the context requires otherwise, throughout this specification and claims, the word "comprise" and variations thereof such as "comprises" and "comprising" are to be interpreted in an open and inclusive sense, i.e., "including but not limited to."

[0031] "Consisting of" means "including, but not limited to," whatever comes before the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are necessary or mandatory, and that no other elements may be present. "Consisting essentially of" means including any elements listed before the phrase, limited to other elements that do not interfere with or contribute to the function or action specified in this disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending on whether they affect the function or action of the listed elements.

[0032] References throughout this specification to "biological activity" or "biological activity" refer to any response induced in an in vitro assay or in a cell, tissue, organ, or organism (e.g., an animal, mammal, or human) as a result of administration of any compound, agent, polypeptide, conjugate, or pharmaceutical composition contemplated herein. Biological activity can refer to agonistic or antagonistic activity. Biological activity can be a beneficial effect; or biological activity can be non-beneficial, i.e., toxic. In some embodiments, biological activity will refer to a positive or negative effect that a drug or pharmaceutical composition has in a living subject, e.g., a mammal, such as a human. Thus, the term "biologically active" is meant to describe any compound that possesses biological activity, as described herein. Biological activity can be assessed by any appropriate means currently known to those of skill in the art. Such assays can be qualitative or quantitative. Those skilled in the art will readily recognize the need to use different assays to assess the activity of different polypeptides; a routine task for the average researcher. Such assays are often easy to perform in a laboratory setting, requiring little optimization, and commercial kits are often available that provide simple, reliable, and reproducible readouts of the biological activity of a wide range of polypeptides, using a variety of techniques common to many laboratories. Where such kits are not available, a researcher of ordinary skill can easily design and optimize an in situ bioactivity assay for a target polypeptide without undue experimentation, since this is a routine aspect of the scientific process.

[0033] Reference to the term "for example" is intended to mean "for example, including but not limited to," so that whatever follows is understood to be merely an example of a particular embodiment, but should in no way be construed as a limiting example. Unless otherwise stated, the use of "for example" is intended to clearly indicate that other embodiments are contemplated and are encompassed by the present invention.

[0034] References throughout this specification to "an embodiment" or "one embodiment" or "an embodiment" or "some embodiments" or "a particular embodiment" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" or "in a particular embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0035] An "increased" or "enhanced" amount is typically a "statistically significant" amount and can include an increase of 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 fold or more (e.g., 100, 500, 1000 fold) (including all integers and decimal points greater than 1 therebetween, e.g., 2.1, 2.2, 2.3, 2.4, etc.) of the amounts or levels described herein. Similarly, a "reduced" or "lower" or "lesser" amount is typically a "statistically significant" amount and can include a decrease of about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more (e.g., 100, 500, 1000 fold) (including all integers and decimal points greater than 1 therebetween, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the amounts or levels described herein.

[0036] The terms "in vitro," "ex vivo," and "in vivo" are intended herein to have their ordinary scientific meaning. Thus, for example, "in "In vitro" is meant to refer to an experiment or reaction that occurs with isolated cellular components, e.g., an enzymatic reaction carried out in a test tube using appropriate substrates, enzymes, donors, and buffers / cofactors as needed. "Ex vivo" is meant to refer to an experiment or reaction that is performed using functional organs or cells that have been removed from an organism or propagated independently of an organism. "In vivo" is meant to refer to an experiment or reaction that occurs within a living organism in its normal, intact state.

[0037] "Mammal" includes both humans and domestic animals, such as laboratory animals and household pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, and rabbits), and non-domestic animals, such as wildlife and the like.

[0038] "Optionally" or "as needed" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not occur.

[0039] A "pharmaceutical composition" refers to a formulation of a compound (e.g., a therapeutically useful polypeptide) and a vehicle generally accepted in the art for delivery of the compound to an animal, e.g., a human. Such a vehicle may therefore include any pharmaceutically acceptable carrier, diluent, or excipient.

[0040] "Pharmaceutically effective excipients" and "pharmaceutically effective carriers" are well known to those skilled in the art, and methods for their preparation will also be readily apparent to those skilled in the art. Such compositions and methods for their preparation can be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995, incorporated herein by reference).

[0041] The terms "polynucleotide," "nucleotide," "nucleotide sequence," and "nucleic acid" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. Non-limiting examples of polynucleotides include: coding or non-coding regions of a gene or gene fragment, locus(s) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can include non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with a labeling component.

[0042] "Subject," as used herein, includes any animal that exhibits a disease or condition or is at risk of exhibiting a disease or condition that can be treated with the agent of the present invention. Suitable subjects include laboratory animals (such as mice, rats, rabbits, or guinea pigs), livestock and domestic animals, or pets (such as cats or dogs). Non-human primates and preferably human patients are also included.

[0043] "Substantially" or "essentially" means an abundant or substantial amount, quantity, or size; almost entirely or completely; for example, 95% or more of a given amount.

[0044] "Therapeutic agent" refers to any compound that, when administered in a therapeutically effective amount to a subject (e.g., preferably a mammal, more preferably a human), can result in treatment of a disease or condition as defined below.

[0045] A "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of a compound of the present invention that, when administered to a subject (e.g., preferably a mammal, more preferably a human), is sufficient to effect treatment of a disease or condition in the animal, as defined below. The amount of a compound of the present invention that constitutes a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, the mode of administration, and the age of the animal to be treated, but can be routinely determined by one of ordinary skill in the art taking into account their own knowledge and this disclosure.

[0046] "Treating" or "treatment," as used herein, encompasses the treatment of a disease or condition of interest in a subject, preferably a human, having the disease or condition, and includes (i) preventing or inhibiting the occurrence of the disease or condition in a subject, particularly if the subject has a predisposition to the condition but has not yet been diagnosed with the condition; (ii) inhibiting the disease or condition, i.e., arresting its onset; (iii) alleviating the disease or condition, i.e., causing regression of the disease or condition; or (iv) alleviating the symptoms caused by the disease or condition. As used herein, the terms "disease," "disorder," and "condition" may be used interchangeably, or may differ in that a particular illness, injury, or condition may not have a known causative agent (such that the etiology has not yet been elucidated) and therefore is not yet recognized as a disorder or disease, but merely as an undesirable condition or syndrome, in which a more or less specific set of symptoms has been identified by a clinician.

[0047] overview The present invention relates, inter alia, to autologous and / or allogeneic B cells that have been modified by the introduction of a nucleic acid to produce follistatin, and to methods of administering the modified B cells (e.g., to treat a disease, disorder, or condition, e.g., a muscular disorder such as muscular dystrophy). In some embodiments, the terms "engineered B cells," "genetically engineered B cells," "modified B cells," and "genetically modified B cells" are used interchangeably herein to refer to B cells that have been modified to contain one or more nucleic acids (e.g., transgenes) to produce follistatin (e.g., a transgene that enables expression of a follistatin polypeptide, such as a therapeutic follistatin polypeptide). Specifically, the modified B cells can be administered as a single dose or multiple doses.

[0048] Thus, the methods for administering the modified B cell compositions described herein are useful for long-term in vivo delivery and expression of follistatin. The present disclosure generally relates to methods for achieving sufficient concentrations and numbers of follistatin-producing cells, and sufficient levels of follistatin in vivo, while ensuring the safety of the product.

[0049] As used herein, the phrases "long-term in vivo survival" and "long-term survival" refer to the survival of modified B cells described herein in a subject for 10 days or longer after administration. Long-term survival can be measured in days, weeks, or even years. In one embodiment, the majority of modified B cells survive in vivo for 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 days or longer after administration. In one embodiment, the majority of the modified B cells survive in vivo for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 weeks or more after administration. In another embodiment, the modified B cells survive in vivo for 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 years or longer. Moreover, while the modified B cells described herein can survive in vivo for 10 days or longer, it is understood that the majority of the modified B cells will survive in vivo for 1, 2, 3, 4, 5, 6, 7, 8, 9 days or longer after administration. Thus, it is contemplated that the modified B cells described herein are useful in short-term treatment (e.g., 4 days) and long-term treatment (e.g., 30 days or longer) methods.

[0050] B cells After leaving the bone marrow, B cells act as antigen-presenting cells (APCs) and internalize antigens. Antigens are taken up and processed by B cells via receptor-mediated endocytosis. Antigens are processed into antigenic peptides, loaded onto MHC II molecules, and presented on the extracellular surface of B cells to CD4+ T helper cells. These T cells bind to MHC II / antigen molecules, leading to B cell activation. After stimulation by T cells, activated B cells begin to differentiate into more specialized cells. Germinal center B cells can differentiate into long-lived memory B cells or plasma cells. Furthermore, secondary immune stimulation can result in memory B cells that give rise to additional plasma cells. The formation of plasma cells from either memory or non-memory B cells is preceded by the formation of precursor plasmablasts, which ultimately differentiate into plasma cells that produce large amounts of antibodies (see, e.g., Trends Immunol. 2009 June; 30(6):277-285; Nature Reviews 2005, 5:231-242). Plasmablasts secrete more antibodies than B cells but less than plasma cells. They divide rapidly, internalize antigens, and continue to present antigens to T cells. Plasmablasts have the ability to migrate to sites of chemokine production (e.g., within the bone marrow), thereby enabling them to differentiate into long-lived plasma cells. Ultimately, plasmablasts can either remain as plasmablasts for several days and then die, or they can differentiate irrevocably into mature, fully differentiated plasma cells. Specifically, plasmablasts that can home to tissues containing plasma cell survival niches (e.g., within the bone marrow) can replace resident plasma cells to become long-lived plasma cells that can continue to secrete high levels of proteins for several years.

[0051] B cells (e.g., to express follistatin) used in the methods described herein include pan-B cells, memory B cells, plasmablasts, and / or plasma cells. In one embodiment, the modified B cells are memory B cells (e.g., modified to express follistatin). In one embodiment, the modified B cells are plasmablasts (e.g., modified to express follistatin). In one embodiment, the modified B cells are plasma cells (e.g., modified to express follistatin).

[0052] Terminally differentiated plasma cells typically do not express common pan-B cell markers such as CD19 and CD20 and express relatively few surface antigens. Because plasma cells express CD38, CD78, CD138, and interleukin-6 receptor (IL-6R) but lack CD45 expression, these markers can be used to identify plasma cells, for example, by flow cytometry. CD27 is also an excellent marker for plasma cells, as naive B cells are CD27-, memory B cells are CD27+, and plasma cells are CD27++. While memory B cell subsets can also express surface IgG, IgM, and IgD, plasma cells do not express these markers on their cell surface. CD38 and CD138 are expressed at high levels on plasma cells (Wikipedia, The Free Encyclopedia. "Plasma cell" page version ID: 404969441; date of last revision: December 30, 2010, 09:54 UTC, retrieved January 4, 2011; Jourdan et al., Blood. December 10, 2009; Volume 114 (Issue 25): 5173-81; Trends Immunol. June 2009; Volume 30 (Issue 6): 277-285; Nature Reviews, 2005, Volume 5: 231-242; Nature Med. 2010, Volume 16: 123-129; Neuberger, MS; Honjo, T.; Alt, Frederick W. (2004). Molecular biology of B cells. Amsterdam: Elsevier, pp. 189-191; Bertil Glader; Greer, John G.; John Foerster; Rodgers, George G.; Paraskevas, Frixos (2008). Wintrobe's Clinical Hematology, Vol. 2, Set. Hagerstwon, MD: Lippincott Williams & Wilkins., p. 347; Walport, Mark; Murphy, Kenneth; Janeway, Charles; Travers, Paul J. (2008). Janeway's immunobiology. New York: Garland Science, pp. 387-388; see also Rawstron AC (May 2006) "Immunophenotyping of plasma cells" Curr Protoc Cytom.

[0053] "Quietant" as used herein refers to a cellular state in which the cells are not actively proliferating.

[0054] "Activated," as used herein, refers to a cellular state in which the cells are actively proliferating and / or producing cytokines in response to a stimulus.

[0055] The terms "differentiate" and "differentiated" as used herein refer to a change in the phenotype of a cell from one cell type or state to another. For example, a memory B cell that transitions into a plasma cell is differentiated.

[0056] The term "subject" is intended to include a living organism in which an adaptive immune response can be elicited (e.g., a mammal). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. In one embodiment, the subject is a human. B cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, tissue from a site of infection, spleen tissue, and tumors. In a preferred embodiment, the source of B cells is PBMCs. Certain embodiments of the present disclosure can use any number of B cell lines available in the art.

[0057] In certain embodiments of the methods described herein, B cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL™ (a copolymer of sucrose and epichlorohydrin that can be used to prepare high-density solutions) separation. In a preferred embodiment, cells from an individual's circulating blood are obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In one embodiment of the methods described herein, the cells are washed with phosphate-buffered saline (PBS). In alternative embodiments, the wash solution can lack calcium, lack magnesium, or lack many, if not all, divalent cations. As one of ordinary skill in the art will readily appreciate, the washing step can be accomplished by methods known to those skilled in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., a Cobe 2991 cell processor) according to the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as PBS. Alternatively, undesirable components of the apheresis sample can be removed and the cells resuspended directly in culture medium.

[0058] B cells can be isolated from peripheral blood or leukapheresis using techniques known in the art. For example, PBMCs can be isolated using FICOLL™ (Sigma-Aldrich, St. Louis, MO), and CD19+ B cells can be purified by negative or positive selection using any of a variety of antibodies known in the art, such as the Rosette tetramer complex system (StemCell Technologies, Vancouver, Canada) or MACS™ MicroBead Technology (Miltenyi Biotec, San Diego, CA). In certain embodiments, memory B cells are isolated as described by Jourdan et al. (Blood. 2009, December 10; vol. 114(25):5173-81). For example, CD19+CD27+ memory B cells can be sorted by FACS after depletion of CD2+ cells using anti-CD2 magnetic beads. Bone marrow plasma cells (BMPCs) can be purified using anti-CD138 magnetic microbead sorting or other similar methods and reagents. Human B cells can be isolated, for example, using CD19 MicroBeads, human (Miltenyi Biotec, San Diego, CA). Human memory B cells can be isolated, for example, using a Memory B Cell Isolation Kit, human (Miltenyi Biotec, San Diego, CA).

[0059] Other isolation kits are commercially available, such as the MagCellect Human B Cell Isolation Kit from R&D Systems (Minneapolis, MN). In certain embodiments, resting B cells can be prepared by sedimentation on a discontinuous Percoll gradient as described in (Defranco et al. (1982) J. Exp. Med. 155:1523).

[0060] In one embodiment, PBMCs are obtained from a blood sample using gradient-based purification (e.g., FICOLL™). In another embodiment, PBMCs are obtained from an apheresis-based collection. In one embodiment, B cells are isolated from PBMCs by isolating pan-B cells. The isolation step can utilize positive and / or negative selection. In one embodiment, negative selection involves depletion of T cells using anti-CD3-conjugated microbeads, thereby providing a T-cell-depleted fraction. In a further embodiment, memory B cells are isolated from the pan-B or T-cell-depleted fraction by positive selection against CD27.

[0061] In one specific embodiment, memory B cells are isolated by depletion of unwanted cells and subsequent positive selection with CD27 MicroBeads. Unwanted cells, such as T cells, NK cells, monocytes, dendritic cells, granulocytes, platelets, and erythroid cells, can be depleted using a cocktail of biotinylated antibodies against CD2, CD14, CD16, CD36, CD43, and CD235a (glycophorin A) and anti-biotin MicroBeads.

[0062] In one embodiment, switched memory B cells are obtained. "Switched memory B cells" or "switched B cells," as used herein, refer to B cells that have undergone isotype class switching. In one embodiment, switched memory B cells are positively selected against IgG. In another embodiment, switched memory B cells are obtained by depleting IgD- and IgM-expressing cells. Switched memory B cells can be isolated, for example, using a Switched Memory B Cell Kit, Human (Miltenyi Biotec, San Diego, CA).

[0063] For example, in one specific embodiment, non-target cells can be labeled with a cocktail of biotinylated CD2, CD14, CD16, CD36, CD43, CD235a (glycophorin A), anti-IgM, and anti-IgD antibodies. Such cells can then be magnetically labeled with anti-biotin MicroBeads. Highly pure switched memory B cells can be obtained by depletion of the magnetically labeled cells.

[0064] In a further embodiment, a promoter sequence from a gene unique to memory B cells, such as the CD27 gene (or other gene specific to memory B cells and not expressed in naive B cells), is used to drive expression of a selectable marker, such as a mutated dihydrofolate reductase that allows for positive selection of memory B cells in the presence of methotrexate. In another embodiment, a promoter sequence from a pan-B cell gene, such as the CD19 gene, is used to drive expression of a selectable marker, such as a mutated dihydrofolate reductase that allows for positive selection of memory B cells in the presence of methotrexate. In another embodiment, T cells are depleted using CD3 or by the addition of cyclosporine. In another embodiment, CD138+ cells are isolated from pan-B cells by positive selection. In yet another embodiment, CD138+ cells are isolated from PBMCs by positive selection. In another embodiment, CD38+ cells are isolated from pan-B cells by positive selection. In yet another embodiment, CD38+ cells are isolated from PBMCs by positive selection. In one embodiment, CD27+ cells are isolated from PBMCs by positive selection. In another embodiment, memory B cells and / or plasma cells are selectively expanded from PBMCs using in vitro culture methods available in the art.

[0065] In vitro B cell culture B cells, such as memory B cells, can be cultured using in vitro methods for activating and differentiating B cells into plasma cells, plasmablasts, or both. As will be appreciated by those skilled in the art, plasma cells can be identified by cell surface protein expression patterns using standard flow cytometry methods. For example, terminally differentiated plasma cells express relatively few surface antigens and do not express common pan-B cell markers such as CD19 and CD20. Instead, plasma cells can be identified by the expression of CD38, CD78, CD138, and IL-6R, and the lack of CD45 expression. CD27 can also be used to identify plasma cells, as naive B cells are CD27-, memory B cells are CD27+, and plasma cells are CD27++. Plasma cells express high levels of CD38 and CD138.

[0066] In one embodiment, the B cells are CD138- memory B cells. In one embodiment, the B cells are CD138+ plasma cells. In one embodiment, the B cells are activated and have a CD138-, CD27+ cell surface phenotype.

[0067] In one embodiment, the B cells are CD20-, CD138- memory B cells. In one embodiment, the B cells are CD20-, CD138+ plasma cells. In one embodiment, the B cells are activated and have a CD20-, CD138-, CD27+ cell surface phenotype.

[0068] In one embodiment, the B cells are CD20-, CD38-, CD138- memory B cells. In one embodiment, the B cells are CD20-, CD38+, CD138+ plasma cells. In one embodiment, the B cells are activated and have a cell surface phenotype of CD20-, CD38-, CD138-, CD27+.

[0069] In one embodiment, B cells are contacted with one or more B cell activators, e.g., any of a variety of cytokines, growth factors, or cell lines known to activate and / or differentiate B cells (see, e.g., Fluckiger et al., Blood 1998, 92:4509-4520; Luo et al., Blood 2009, 113:1422-1431). Such factors include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15 , IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL- 30, IL-31, IL-32, IL-33, IL-34 and IL-35, IFN-γ, IFN-α, IFN-β, IFN-δ, C-type chemokines XCL1 and XCL2, CC-type chemokines (currently including CCL1 to CCL28) and CXC-type chemokines (currently including CXCL1 to CXCL17), and members of the TNF superfamily (e.g., TNF-α, 4-1 The antibody may be selected from the group consisting of, but is not limited to, BB ligand, B cell activating factor (BLyS), FAS ligand, sCD40L (including multimeric versions of sCD40L; for example, histidine-tagged soluble recombinant CD40L in combination with an anti-poly-histidine mAb to group multiple sCD40L molecules together), lymphotoxin, OX40L, RANKL, TRAIL), CpG, and other toll-like receptor agonists (e.g., CpG).

[0070] B cell activating factors can be added to in vitro cell cultures at various concentrations to achieve a desired outcome (e.g., expansion or differentiation). In one embodiment, a B cell activating factor is utilized in the expansion of B cells in culture. In one embodiment, a B cell activating factor is utilized in the differentiation of B cells in culture. In another embodiment, a B cell activating factor is utilized in both the expansion and differentiation of B cells in culture. In one embodiment, a B cell activating factor is provided at the same concentration for expansion and differentiation. In another embodiment, a B cell activating factor is provided at a first concentration for expansion and a second concentration for differentiation. It is contemplated that a B cell activating factor can be utilized 1) in the expansion of B cells but not in the differentiation of B cells, 2) in the differentiation of B cells but not in the expansion of B cells, or 3) in the expansion and differentiation of B cells.

[0071] For example, in some embodiments, B cells are cultured with a B cell culture medium containing one or more B cell activating factors selected from CD40L, IL-2, IL-4, and IL-10 for B cell expansion. In one embodiment, B cells are cultured with 0.25 to 5.0 μg / ml CD40L. In one embodiment, the concentration of CD40L is 0.5 μg / ml. In one embodiment, a cross-linking agent (such as an anti-HIS antibody in combination with HIS-tagged CD40L) is used to generate CD40L multimers. In one embodiment, CD40L molecules are covalently linked or held together using a protein multimerization domain (e.g., the Fc region of IgG or a leucine zipper domain). In one embodiment, CD40L is conjugated to beads. In one embodiment, CD40L is expressed from feeder cells. In one embodiment, B cells are cultured with 1 to 10 ng / ml IL-2. In one embodiment, the concentration of IL-2 is 5 ng / ml. In one embodiment, the B cells are cultured with 1 to 10 ng / ml IL-4. In one embodiment, the concentration of IL-4 is 2 ng / ml. In one embodiment, the B cells are cultured with 10 to 100 ng / ml IL-10. In one embodiment, the concentration of IL-10 is 40 ng / ml.

[0072] In one embodiment, B cells are cultured with a B cell culture medium containing one or more B cell activating factors selected from CD40L, IL-2, IL-4, IL-10, IL-15, and IL-21 for B cell expansion. In one embodiment, B cells are cultured with 0.25 to 5.0 μg / ml CD40L. In one embodiment, the concentration of CD40L is 0.5 μg / ml. In one embodiment, a cross-linking agent (such as an anti-HIS antibody in combination with HIS-tagged CD40L) is used to generate CD40L multimers. In one embodiment, CD40L molecules are covalently linked or held together using a protein multimerization domain (e.g., the Fc region of IgG or a leucine zipper domain). In one embodiment, CD40L is conjugated to beads. In one embodiment, CD40L is expressed from feeder cells. In one embodiment, B cells are cultured with 1 to 10 ng / ml IL-2. In one embodiment, the concentration of IL-2 is 5 ng / ml. In one embodiment, B cells are cultured with 1 to 10 ng / ml IL-4. In one embodiment, the concentration of IL-4 is 2 ng / ml. In one embodiment, B cells are cultured with 10 to 100 ng / ml IL-10. In one embodiment, the concentration of IL-10 is 40 ng / ml. In one embodiment, B cells are cultured with 50 to 150 ng / ml IL-15. In one embodiment, the concentration of IL-15 is 100 ng / ml. In one embodiment, B cells are cultured with 50 to 150 ng / ml IL-21. In one embodiment, the concentration of IL-21 is 100 ng / ml. In a specific embodiment, B cells are cultured with a B cell culture medium containing CD40L, IL-2, IL-4, IL-10, IL-15, and IL-21 for B cell expansion.

[0073] For example, in one embodiment, B cells are cultured with B cell culture medium containing B cell activators CD40L, IL-2, IL-4, IL-10, IL-15, and IL-21 for B cell expansion, and CD40L is crosslinked with a crosslinking agent to create CD40L multimers. Such a culture system can be maintained throughout the culture period (e.g., a 7-day culture period), and the B cells are transfected or otherwise engineered to express a transgene of interest (e.g., an exogenous polypeptide such as FST). The transgene can be integrated into the B cells (e.g., by a viral or non-viral vector). The transgene can be expressed in the B cells by using a transposon. The transgene can be expressed in the B cells for targeted integration of the transgene into the genome of the B cells. Targeted integration can be by homologous recombination. Homologous recombination can occur upon a nuclease-induced double-strand break. The nuclease can be, for example, a zinc finger nuclease, a TALE-nuclease (TALEN), a meganuclease (e.g., a homing endonuclease), or can be from a CRISPR / CAS9-nuclease system.

[0074] In another example, in some embodiments, B cells are cultured with a B cell culture medium containing one or more B cell activating factors selected from CD40L, IFN-α, IL-2, IL-6, IL-10, IL-15, IL-21, and P-class CpG oligodeoxynucleotides (p-ODNs) for B cell differentiation. In one embodiment, B cells are cultured with 25 to 75 ng / ml CD40L. In one embodiment, the concentration of CD40L is 50 ng / ml. In one embodiment, B cells are cultured with 250 to 750 U / ml IFN-α. In one embodiment, the concentration of IFN-α is 500 U / ml. In one embodiment, B cells are cultured with 5 to 50 U / ml IL-2. In one embodiment, the concentration of IL-2 is 20 U / ml. In one embodiment, B cells are cultured with 25 to 75 ng / ml IL-6. In one embodiment, the concentration of IL-6 is 50 ng / ml. In one embodiment, B cells are cultured with 10 to 100 ng / ml IL-10. In one embodiment, the concentration of IL-10 is 50 ng / ml. In one embodiment, B cells are cultured with 1 to 20 ng / ml IL-15. In one embodiment, the concentration of IL-15 is 10 ng / ml. In one embodiment, B cells are cultured with 10 to 100 ng / ml IL-21. In one embodiment, the concentration of IL-21 is 50 ng / ml. In one embodiment, B cells are cultured with 1 to 50 μg / ml p-ODN. In one embodiment, the concentration of p-ODN is 10 μg / ml.

[0075] In one embodiment, B cells are contacted or cultured on feeder cells. In one embodiment, the feeder cells are a stromal cell line, e.g., mouse stromal cell line S17 or MS5. In another embodiment, isolated CD19+ cells are cultured with one or more B cell activating cytokines, such as IL-10 and IL-4, in the presence of fibroblasts expressing CD40-ligand (CD40L, CD154). In one embodiment, CD40L is provided bound to a surface, such as a tissue culture plate or beads. In another embodiment, purified B cells are cultured with CD40L and one or more cytokines or factors selected from IL-10, IL-4, IL-7, p-ODN, CpG DNA, IL-2, IL-15, IL6, and IFN-α, in the presence or absence of feeder cells.

[0076] In another embodiment, B cell activating factors are provided by transfection into B cells or other feeder cells. In this context, one or more factors that promote the differentiation of B cells into antibody-secreting cells and / or the longevity of antibody-producing cells can be used. Such factors include, for example, anti-apoptotic factors such as Blimp-1, TRF4, Bcl-x1, or Bcl5, or constitutively active variants of the CD40 receptor. Furthermore, factors that promote the expression of downstream signaling molecules, such as TNF receptor-associated factors (TRAFs), can also be used in B cell activation / differentiation. In this regard, the cell activation, cell survival, and anti-apoptotic functions of the TNF receptor superfamily are largely mediated by TRAFs 1-6 (see, e.g., R.H. Arch et al., Genes Dev. 12 (1998) pp. 2821-2830). Downstream effectors of TRAF signaling include transcription factors in the NF-κB and AP-1 families, which can turn on genes involved in various aspects of cellular and immune function. Furthermore, activation of NF-κB and AP-1 has been shown to result in cellular protection from apoptosis through the transcription of anti-apoptotic genes.

[0077] In another embodiment, proteins derived from Epstein-Barr virus (EBV) are used to activate and / or differentiate B cells or promote the longevity of antibody-producing cells, including, but not limited to, EBNA-1, EBNA-2, EBNA-3, LMP-1, LMP-2, EBER, miRNA, EBV-EA, EBV-MA, EBV-VCA, and EBV-AN.

[0078] In certain embodiments, contacting B cells with a B cell activator using the methods provided herein results in, among other things, cell proliferation (i.e., expansion), modulation of the IgM+ cell surface phenotype to a phenotype consistent with activated mature B cells, Ig secretion, and isotype switching. CD19+ B cells can be isolated using known and commercially available cell separation kits, such as the MiniMACS™ Cell Separation System (Miltenyi Biotech, Bergisch Gladbach, Germany). In certain embodiments, CD40L fibroblasts are irradiated prior to use in the methods described herein. In one embodiment, B cells are cultured in the presence of one or more of IL-3, IL-7, Flt3 ligand, thrombopoietin, SCF, IL-2, IL-10, G-CSF, and CpG. In certain embodiments, the method comprises culturing B cells in the presence of one or more of the above-mentioned factors in conjunction with transformed stromal cells (e.g., MS5) that provide low levels of tethered CD40L and / or plate- or bead-bound CD40L.

[0079] As described above, B cell activators induce the expansion, proliferation, or differentiation of B cells. Thus, B cells are contacted with one or more B cell activators listed above to obtain an expanded cell population. The cell population can be expanded before transfection. Alternatively, or in addition, the cell population can be expanded after transfection. In one embodiment, the expansion of the B cell population comprises culturing the cells with IL-2, IL-4, IL-10, and CD40L (see, e.g., Neron et al., PLoS ONE, 2012, 7(12):e51946). In one embodiment, the expansion of the B cell population comprises culturing the cells with IL-2, IL-10, CpG, and CD40L. In one embodiment, the expansion of the B cell population comprises culturing the cells with IL-2, IL-4, IL-10, IL-15, IL-21, and CD40L. In one embodiment, expansion of the B cell population comprises culturing the cells with IL-2, IL-4, IL-10, IL-15, IL-21 and multimerized CD40L.

[0080] In another embodiment, the expansion of a B cell population is induced and / or enhanced by a transgene introduced into the B cells. For example, B cells containing a recombinant or engineered receptor that induces a cell signaling pathway (e.g., signaling downstream of CD40) upon binding to its ligand (e.g., a soluble or cell surface-expressed ligand). In one embodiment, the B cells overexpress CD40 due to expression of a CD40 transgene. In another embodiment, the B cells express an engineered receptor, including, for example, a recombinantly engineered antibody. In one embodiment, the engineered receptor is similar to a chimeric antigen receptor (CAR) and comprises a fusion protein of an scFv and the intracellular signaling portion of a B cell receptor (e.g., CD40).

[0081] In one embodiment, the expansion of B cell populations is induced and / or enhanced by small molecule compounds added to the cell culture. For example, compounds that bind to and dimerize CD40 can be used to induce the CD40 signaling pathway.

[0082] As known to those skilled in the art, any of a variety of culture media can be used in the present methods (see, e.g., Current Protocols in Cell Culture, 2000-2009, John Wiley & Sons, Inc.). In one embodiment, media for use in the methods described herein include, but are not limited to, Iscove's Modified Dulbecco's Medium (with or without fetal bovine or other appropriate serum). Exemplary media also include, but are not limited to, IMDM, RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20. In further embodiments, the media can contain additives such as detergents, antibodies, plasmanate or reducing agents (e.g., N-acetyl-cysteine, 2-mercaptoethanol), one or more antibiotics, and / or insulin, transferrin, sodium selenite, and cyclosporine. In some embodiments, IL-6, soluble CD40L, and cross-linking enhancers can also be used.

[0083] B cells are cultured under conditions and for a sufficient period of time to achieve the desired differentiation and / or activation. In certain embodiments, B cells are cultured under conditions and for a sufficient period of time such that 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% of the B cells are differentiated and / or activated as desired. In one embodiment, B cells are activated and differentiated into a mixed population of plasmablasts and plasma cells. As will be recognized by those skilled in the art, plasmablasts and plasma cells can be differentiated into a mixed population of plasmablasts and plasma cells, including those expressing CD38, CD78, IL-6R, CD27, and / or CD47. highand CD138, and / or lack or reduced expression of one or more of CD19, CD20, and CD45, can be identified using standard flow cytometry methods as described elsewhere herein. As will be appreciated by those skilled in the art, memory B cells are generally CD20+, CD19+, CD27+, CD38-, while early plasmablasts are CD20-, CD19+, CD27++, CD38++. In one embodiment, cells cultured using the methods described herein are CD20-, CD38+, CD138-. In another embodiment, the cells have a CD20-, CD38+, CD138+ phenotype. In certain embodiments, the cells are cultured for 1-7 days. In further embodiments, the cells are cultured for 7, 14, 21 days, or longer. Thus, cells can be cultured under appropriate conditions for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 days or longer. Cells can be replated and medium and supplements can be added or replaced as needed using techniques known in the art.

[0084] In certain embodiments, the B cells are cultured under conditions and for a sufficient period of time such that at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells are differentiated and activated to produce Ig and / or express the transgene.

[0085] Induction of B cell activation is mediated by RNA 3 By techniques such as 3H-uridine incorporation (RNA synthesis increases as B cells differentiate) or by measuring DNA synthesis as cells proliferate 3It can be measured by H-thymidine incorporation. In one embodiment, interleukin-4 (IL-4) can be added to the culture medium at a concentration appropriate for enhancing B cell proliferation (e.g., about 10 ng / ml).

[0086] Alternatively, B cell activation can be measured as a function of immunoglobulin secretion. For example, CD40L can be added to resting B cells along with IL-4 (e.g., 10 ng / ml) and IL-5 (e.g., 5 ng / ml), or other cytokines that activate B cells. Flow cytometry can also be used to measure cell surface markers typical of activated B cells. See, e.g., Civin CI, Loken MR, Int'l J. Cell Cloning 987; vol. 5: 1-16; Loken, MR et al. Flow Cytometry Characterization of Erythroid, Lymphoid and Mononomyeloid Lineages See in Normal Human Bone Marrow, in Flow Cytometry in Hematology, Laerum OD, Bjerksnes R., eds., Academic Press, New York 1992; 31-42; and LeBein TW et al., Leukemia 1990; 4:354-358.

[0087] After culturing for an appropriate period of time, such as 2, 3, 4, 5, 6, 7, 8, 9 days or more, generally around 3 days, additional volumes of culture medium can be added. Supernatants from individual cultures can be collected at various time points in culture and quantified for IgM and IgG1 as described in Noelle et al. (1991) J. Immunol. 146:1118-1124. In one embodiment, cultures are harvested and measured for expression of the transgene of interest using flow cytometry, enzyme-linked immunosorbent assay (ELISA), ELISPOT, or other assays known in the art.

[0088] In another embodiment, ELISA is used to measure antibody isotype production, e.g., IgM, or the product of the transgene of interest. In certain embodiments, IgG determination is performed using a commercially available antibody, such as goat anti-human IgG, as a capture antibody, followed by detection using any of a variety of appropriate detection reagents, such as biotinylated goat anti-human Ig, streptavidin alkaline phosphatase, and substrate.

[0089] In certain embodiments, B cells are cultured under conditions and for a sufficient period of time such that the number of cells is 1, 10, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more times the number of B cells at the initiation of culture. In one embodiment, the number of cells is 10 to 1000 times (including consecutive integers therein) the number of B cells at the initiation of culture. For example, the expanded B cell population is at least 10 times the size of the initially isolated B cell population. In another embodiment, the expanded B cell population is at least 100 times the size of the initially isolated B cell population. In one embodiment, the expanded B cell population is at least 500 times the size of the initially isolated B cell population.

[0090] B cell manipulation In various embodiments, the present disclosure provides methods for transfecting, infecting, or otherwise incorporating a transgene (e.g., a follistatin transgene) into B cells such that the transgene is expressed in the B cells. Any of the integration methods described herein or known in the art can, in some embodiments, be utilized to express follistatin in B cells.

[0091] In one embodiment, the genetically modified B cells are transfected with a transgene. In a specific embodiment, the genetically modified B cells are with a follistatin transgene.

[0092] Exemplary methods for transfecting cells are provided in WO2014 / 152832 and WO2016 / 100932, both of which are incorporated herein by reference in their entirety. Transfection of B cells can be achieved using any of a variety of methods available in the art for introducing DNA or RNA into B cells. Suitable techniques include calcium phosphate transfection, DEAE-dextran, electroporation, pressure-mediated transfection or "cell squeezing" (e.g., CellSqueeze microfluidic system, SQZ Biotechnologies), nanoparticle- or liposome-mediated transfection, and transduction using retroviruses or other viruses, such as vaccinia. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratories; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197; US 5,124,259; US 5,297,983; US 5,283,185; US 5,661,018; US 6,878,548; US 7,799,555; US 8,551,780; and US 8,633,029. An example of a commercially available electroporation technique suitable for B cells is the Nucleofector™ transfection technique.

[0093] Transfection can be performed before or during in vitro culture of isolated B cells in the presence of one or more of the above-mentioned activation and / or differentiation factors. For example, the cells are transfected on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 of in vitro culture. In one embodiment, the cells are transfected on day 1, 2, or 3 of in vitro culture. In a specific embodiment, the cells are transfected on day 2. For example, the cells are electroporated on day 2 of in vitro culture, for example, to deliver a plasmid, transposon, minicircle, or self-replicating RNA. In another embodiment, the cells are transfected on day 4, 5, 6, or 7 of in vitro culture. In a specific embodiment, the cells are transfected on day 6 of in vitro culture. In another embodiment, the cells are transfected on day 5 of in vitro culture.

[0094] In one embodiment, the cells are transfected or otherwise engineered (e.g., by targeted integration of a follistatin transgene) prior to activation. In another embodiment, the cells are transfected or otherwise engineered (e.g., by targeted integration of a follistatin transgene) during activation. In one embodiment, the cells are transfected or otherwise engineered (e.g., by targeted integration of a follistatin transgene) after activation. In one embodiment, the cells are transfected or otherwise engineered (e.g., by targeted integration of a follistatin transgene) prior to differentiation. In another embodiment, the cells are transfected or otherwise engineered (e.g., by targeted integration of a follistatin transgene) during differentiation. In one embodiment, the cells are transfected or otherwise engineered (e.g., by targeted integration of a follistatin transgene) after differentiation.

[0095] In one embodiment, non-viral vectors are used to deliver DNA or RNA (e.g., DNA or RNA comprising a sequence encoding a follistatin polypeptide) to memory B cells and / or plasma cells. For example, systems that can facilitate transfection of memory B cells and / or plasma cells without the need for a viral integration system include, but are not limited to, transposons (e.g., Sleeping Beauty or other transposon systems such as Piggybac), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPRs), meganucleases, minicircles, replicons, artificial chromosomes (e.g., bacterial artificial chromosomes, mammalian artificial chromosomes, and yeast artificial chromosomes), plasmids, cosmids, and bacteriophages.

[0096] In some embodiments, such non-viral vector systems can also be delivered by viral vectors known in the art or described below. For example, in some embodiments, viral vectors (e.g., retroviruses, lentiviruses, adenoviruses, adeno-associated viruses) are utilized to deliver one or more non-viral vectors (e.g., one or more of the above-mentioned zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR) meganucleases, etc.), or any other enzyme / complementary vector, polynucleotide, and / or polypeptide that can facilitate targeted integration. Thus, in some embodiments, cells (e.g., B cells, such as memory B cells and / or plasma cells) can be engineered to express an exogenous sequence (e.g., a sequence encoding a follistatin polypeptide) via targeted integration methods. Such methods are known in the art and can include cleaving an endogenous locus in a cell using one or more nucleases (e.g., ZFNs, TALENs, CRISPR / Cas, meganucleases) and administering a follistatin transgene to the cell such that it is integrated into the endogenous locus and expressed in the cell. The follistatin transgene can be contained in a donor sequence that is integrated into the host cell's DNA at or near the point of cleavage by the nuclease.

[0097] Integration of an exogenous sequence (e.g., a sequence encoding a follistatin polypeptide) can occur by recombination. As will be apparent to those skilled in the art, "recombination" refers to the process of exchanging genetic information between two polynucleotides, including, but not limited to, donor capture by non-homologous end joining (NHEJ) and homologous recombination. Recombination can be homologous recombination. For purposes of this disclosure, "homologous recombination (HR)" refers to a specialized form of such exchange that occurs, for example, during repair of a double-strand break in a cell by a homology-directed repair mechanism. This process utilizes nucleotide sequence homology, whereby a "donor" molecule (e.g., a donor polynucleotide sequence or a donor vector containing such a sequence) is used by the cell's DNA repair machinery as a template to repair a "target" molecule (i.e., a molecule that has experienced a double-strand break), and these means result in the transfer of genetic information from the donor to the target. In some embodiments of HR-directed integration, the donor molecule can contain at least two regions of homology to the genome ("homology arms"). In some embodiments, the homology arms can be, for example, at least 50-100 base pairs in length. The homology arms can have substantial DNA homology to a region of genomic DNA adjacent to the cleavage site where targeted integration is to occur. The homology arms of the donor molecule can flank the DNA (e.g., including DNA encoding follistatin) to be integrated into the target genome or target DNA locus. Chromosomal cleavage followed by repair using the homologous region of the plasmid DNA as a template can result in the transfer of the intervening transgene flanked by the homology arms into the genome. See, e.g., Koller et al. (1989) Proc. Nat'l. Acad. Sci. USA 86(22):8927-8931; Thomas et al. (1986) Cell 44(3):419-428. The frequency of this type of homology-directed targeted integration can be increased up to 10 by deliberately creating a double-strand break near the target region. 527(9):851-857; Lombardo et al. (2007) Nature Biotech. 25(11):1298-1306; Moehle et al. (2007) Proc. Nat'l Acad. Sci. USA 104(9):3055-3060; Rouet et al. (1994) Proc. Nat'l Acad. Sci. USA 91(13):6064-6068.

[0098] Any nuclease capable of mediating targeted cleavage of a genomic locus such that a transgene (e.g., a follistatin transgene) can be integrated into the genome of the target cell (e.g., by recombination, such as HR) can be utilized in engineering cells (e.g., memory B cells or plasmablasts) in accordance with the present disclosure.

[0099] Double-strand breaks (DSBs) or nicks can be generated by site-specific nucleases such as zinc finger nucleases (ZFNs), TAL effector domain nucleases (TALENs), meganucleases, or using the CRISPR / Cas9 system with engineered crRNA / tract RNA (single guide RNA) to guide specific cleavage. See, for example, Burgess (2013) Nature Reviews Genetics 14:80-81; Urnov et al. (2010) Nature 14:80-81; the disclosures of which are incorporated herein by reference in their entireties for all purposes. 435(7042):646-51; U.S. Patent Application Publication Nos. 20030232410; 20050208489; 20050026157; 20050064474; 20060188987; 20090263900; 20090117617; 20100047805; 20110207221; 20110301073 and International Publication No. WO2007 / 014275.

[0100] In some embodiments, cells (e.g., memory B cells or plasmablasts) are engineered by zinc finger nuclease-mediated targeted integration of a donor construct (e.g., a follistatin donor construct). Zinc finger nucleases (ZFNs) are enzymes that can recognize and cleave target nucleotide sequences with specificity due to the coupling of a "zinc finger DNA-binding protein" (ZFP) (or binding domain), which binds to DNA in a sequence-specific manner via one or more zinc fingers and a nuclease enzyme. ZFNs can include any suitable cleavage domain (e.g., a nuclease enzyme) operably linked to the ZFP DNA-binding domain to form engineered ZFNs that can facilitate site-specific cleavage of target DNA sequences (see, e.g., Kim et al. (1996) Proc Natl Acad Sci USA 93(3):1156-1160). For example, the ZFN can include a target-specific ZFP linked to a FOK1 enzyme or a portion of a FOK1 enzyme. In some embodiments, the ZFN used in ZFN-mediated targeted integration approach utilizes two separate molecules, each comprising a subunit of FOK1 enzyme bound to a ZFP, each ZFP having specificity for the DNA sequence adjacent to the target cleavage site, and when these two ZFPs bind to their respective target DNA sites, the FOK1 enzyme subunits are brought into close proximity with each other, bind together, and activate the nuclease activity of cleaving the target cleavage site.ZFN has been used for genome modification in various organisms (for example, U.S. Patent Application Publication No. 20030232410; U.S. Patent Application Publication No. 20050208489; U.S. Patent Application Publication No. 20050026157; U.S. Patent Application Publication No. 20050064474; U.S. Patent Application Publication No. 20060188987; U.S. Patent Application Publication No. 20060063231; and International Publication No. WO07 / 014,275, the entire contents of which are incorporated herein by reference). Custom ZFPs and ZFNs are commercially available, for example, from Sigma Aldrich (St. Louis, MO), and such custom ZFNs can also be used to routinely target and cleave any location in DNA.

[0101] In some embodiments, cells (e.g., memory B cells or plasmablasts) are engineered by CRISPR / Cas (e.g., CRISPR Cas9) nuclease-mediated integration of a donor construct (e.g., a follistatin donor construct). The CRISPR (clustered regularly interspaced short palindromic repeats) / Cas (CRISPR-associated) nuclease system is an engineered nuclease system based on a bacterial system that can be used for genome engineering. It is based in part on the adaptive immune response of many bacteria and archaea. When a virus or plasmid invades a bacterium, a segment of the invader's DNA is converted into CRISPR RNA (crRNA) by the "immune" response. This crRNA then associates with another type of RNA, called tracrRNA, through a region of partial complementarity to guide the Cas9 nuclease to a region of homology to the crRNA in the target DNA, called the "protospacer." Cas9 cleaves DNA to generate blunt ends at DSBs at sites specified by a 20-nucleotide guide sequence contained within the crRNA transcript. Cas9 requires both the crRNA and tracrRNA for site-specific DNA recognition and cleavage. This system has now been engineered to combine the crRNA and tracrRNA into a single molecule (a "single guide RNA"), and the portion of the single guide RNA corresponding to the crRNA can be engineered to guide the Cas9 nuclease to target any desired sequence (Jinek et al. (2012) Science 337:816-821; Jinek et al. (2013) eLife 2:e00471; and David Segal (2013) eLife (See Vol. 2:e00563.) Thus, the CRISPR / Cas system can be engineered to create DSBs at desired targets in the genome, and repair of the DSBs can be affected by the use of repair inhibitors, which cause an increase in error-prone repair. As will be apparent to those skilled in the art, in addition to Cas9, other CRISPR nucleases are known and suitable for use in the present invention.

[0102] In some embodiments, CRISPR / Cas nuclease-mediated integration utilizes type II CRISPR. Type II CRISPR is one of the best-characterized systems and performs targeted DNA double-strand breaks in four sequential steps. First, two non-coding RNAs, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat region of the pre-crRNA and mediates processing of the pre-crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to the target DNA through Watson-Crick base pairing between the spacer in the crRNA and the protospacer in the target DNA adjacent to the protospacer adjacent motif (PAM), an additional requirement for target recognition. Fourth, Cas9 mediates cleavage of the target DNA to create a double-strand break within the protospacer.

[0103] The Cas9-related CRISPR / Cas system comprises two non-coding RNA components: the tracrRNA and a pre-crRNA array containing nuclease guide sequences (spacers) interspersed with identical direct repeats (DRs). To achieve genome engineering using the CRISPR / Cas system, both of these RNA functions must be present (see Cong et al. (2013) Sciencexpress 1 / 10.1126 / science 1231143). In some embodiments, the tracrRNA and pre-crRNA are provided by separate expression constructs or as separate RNAs. In other embodiments, chimeric RNAs are constructed in which an engineered mature crRNA (which confers target specificity) is fused to the tracrRNA (which provides interaction with Cas9) to create a chimeric cr-RNA-tracrRNA hybrid (also designated a single guide RNA). (See Jinek, supra, and Cong, supra).

[0104] In some embodiments, a single guide RNA containing both crRNA and tracrRNA can be engineered to guide the Cas9 nuclease to target any desired sequence (e.g., Jinek et al. (2012) Science 337:816-821; Jinek et al. (2013) eLife 2:e00471; David Segal (2013) eLife 2:e00563). Thus, the CRISPR / Cas system can be engineered to create DSBs at desired targets in the genome.

[0105] Custom CRISPR / Cas systems are commercially available, for example, from Dharmacon (Lafayette, CO), and such custom single-guide RNA sequences can also be used to routinely target and cleave any location in DNA. Single-stranded DNA templates for recombination can be synthesized (e.g., by oligonucleotide synthesis methods known in the art and commercially available) or can be provided in a vector, for example, a viral vector such as AAV.

[0106] In some embodiments, cells (e.g., memory B cells or plasmablasts) are engineered by TALE-nuclease (TALEN)-mediated targeted integration of a donor construct (e.g., a follistatin donor construct). A "TALE DNA-binding domain" or "TALE" is a polypeptide containing one or more TALE repeat domains / units. The repeat domain is responsible for the 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 to other TALE repeat sequences in naturally occurring TALE proteins. TAL-effectors can contain tandemly repeated nuclear localization sequences, acidic transcriptional activation domains, and centralizing domains, with each repeat containing approximately 34 amino acids that are key to the DNA-binding specificity of such proteins. (See, e.g., Schornack S, et al. (2006) J Plant Physiol 163(3):256-272). TAL effectors rely on sequences found in tandem repeats containing approximately 102 bp, and the repeats are typically 91-100% homologous to each other (e.g., Bonas et al. (1989) MoI Gen Genet 218:127-136). Such DNA-binding repeats can be engineered to place new combinations and numbers of repeats into proteins to generate artificial transcription factors that can interact with new sequences and activate expression of non-endogenous reporter genes (e.g., Bonas et al. (1989) MoI Gen Genet 218:127-136). Genet 218:127-136). Engineered TAL proteins can be linked to a FokI cleavage half-domain to generate TAL effector domain nuclease fusions (TALENs) for cleaving target-specific DNA sequences (e.g., Christian et al. (2010) Genetics, epub 10.1534 / genetics.110.120717).

[0107] Custom TALENs are commercially available, for example, from Thermo Fisher Scientific (Waltham, Mass.), and can also be used to routinely target and cleave any location in DNA.

[0108] In some embodiments, cells (e.g., memory B cells or plasmablasts) are engineered by meganuclease-mediated targeted integration of a donor construct (e.g., a follistatin donor construct). Meganucleases (or "homing endonucleases") are endonucleases that bind to and cleave double-stranded DNA at recognition sequences of greater than 12 base pairs. Naturally occurring meganucleases can be monomeric (e.g., I-SceI) or dimeric (e.g., I-CreI). Naturally occurring meganucleases recognize 15-40 base pair cleavage sites and are generally grouped into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cyst box family, and the HNH family. Exemplary homing endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII, and I-TevIII, whose recognition sequences are known. See also U.S. Patent No. 5,420,032; U.S. Patent No. 6,833,252; Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler et al. (1994) Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al. (1996) J. Mol. Biol. 263:163-180; Argast et al. (1998) J. Mol. Biol. 280:345-353 and the New England Biolabs catalog. The term "meganuclease" includes monomeric meganucleases, dimeric meganucleases, and monomers that associate to form dimeric meganucleases.

[0109] In certain embodiments, the methods and compositions described herein utilize nucleases, including engineered (non-naturally occurring) homing endonucleases (meganucleases). Recognition sequences for homing endonucleases and meganucleases are known, such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII, and I-TevIII. See also U.S. Patent No. 5,420,032; U.S. Patent No. 6,833,252; Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler et al. (1994) Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al. (1996) J. Mol. Biol. 263:163-180; Argast et al. (1998) J. Mol. Biol. 280:345-353 and the New England Biolabs catalog. In addition, the DNA binding specificity of homing endonucleases and meganucleases can be engineered to bind to non-natural target sites. See, for example, Chevalier et al. (2002) Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic Acids Res. 31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques et al. (2007) Current Gene Therapy 7:49-66; U.S. Patent Application Publication No. 20070117128. The DNA binding domain of homing endonucleases and meganucleases can be altered in the context of the nuclease as a whole (i.e., so that the nuclease contains a cognate cleavage domain) or fused to a heterologous cleavage domain.Custom meganucleases are commercially available, for example, from New England Biolabs (Ipswich, Mass.), and can also be routinely targeted and cleaved at any location in DNA.

[0110] Engineering B cells can include administering one or more nucleases (e.g., ZFN, TALEN, CRISPR / Cas, meganucleases) to the B cells, for example, by one or more vectors encoding the nucleases, such that the vectors containing the encoded nucleases are taken up by the B cells. The vectors can be viral vectors.

[0111] In some embodiments, a nuclease cleaves a specific endogenous locus (e.g., a safe harbor gene or locus of interest) in a cell (e.g., a memory B cell or a plasma cell), and one or more exogenous (donor) sequences (e.g., transgenes) are administered (e.g., one or more vectors containing these exogenous sequences). In such embodiments, the donor sequences may encode follistatin (e.g., a follistatin transgene). The nuclease can induce a double-strand break (DSB) or a single-strand break (nick) in the target DNA. In some embodiments, targeted insertion of the donor transgene (e.g., a follistatin donor transgene) can be achieved by homology-directed repair (HDR), non-homologous repair mechanisms (e.g., NHEJ-mediated end-capture), or insertion and / or deletion of nucleotides (e.g., endogenous sequences) at the site of integration of the transgene (e.g., follistatin transgene) into the genome of the cell. In one embodiment, the method of transfecting B cells comprises electroporating the B cells prior to contacting the B cells with the vector. In one embodiment, the cells are electroporated on a day ranging from day 1 to day 12 of in vitro culture. In one embodiment, the cells are electroporated on day 1, 2, 3, 4, 5, 6, 7, 8, or 9 of in vitro culture. In one embodiment, the cells are electroporated on day 2 of in vitro culture for delivery of the plasmid.

[0112] In one embodiment, cells are transfected using a transposon. As used herein, the term "transposonization" may refer, in some embodiments, to cells that are transfected using a transposon. Numerous transposon systems are known in the art and suitable for use in the present invention. For example, the Sleeping Beauty transposon system and the Piggybac transposon system are well known in the art and suitable for use in the present invention. See, for example, Hackett PB, et al., Evaluating Risks, 2004, pp. 111-114, 2004, each of which is incorporated herein by reference in its entirety. of Insertional Mutagenesis by DNA Transposons in Gene Therapy, Transl. Res. 2013 April; 161(4): 265-283; Hudecek M, et al., Going non-viral: the Sleeping Beauty transposon system breaks on through to the clinical side, Crit Rev Biochem Mol Biol. 2017 Aug;52(4):355-380. In some embodiments, the cells are transfected using a Sleeping Beauty transposon. The Sleeping Beauty transposon, in some embodiments, may be a T2 Sleeping Beauty transposon or a T4 Sleeping Beauty transposon. In some embodiments, utilizing the Sleeping Beauty transposon system may include transfecting B cells (e.g., by electroporation) with a DNA construct encoding the transposon system machinery and a DNA construct encoding a follistatin polypeptide. In some embodiments, the DNA construct encoding the transposon system machinery may be pCMV-SB100x. In some embodiments, utilizing the Sleeping Beauty transposon system includes transfecting B cells (e.g., by electroporation) with a DNA construct encoding a follistatin polypeptide and further transfecting the B cells with mRNA encoding the transposon system machinery. In some embodiments, the mRNA encoding the transposon system machinery encodes the SB100x transposase. In some embodiments, the cells are transfected using a Piggybac transposon. In one embodiment, the cells are transfected using a transposon (e.g., a T2 or T4 Sleeping Beauty transposon or a Piggybac transposon) on days ranging from 1 to 12 of in vitro culture. In one embodiment, the cells are transfected using a transposon (e.g., a T2 or T4 Sleeping Beauty transposon or a Piggybac transposon) on days 1, 2, 3, 4, 5, 6, 7, 8, or 9 of in vitro culture. In one embodiment, the cells are transfected using a minicircle on days ranging from 1 to 12 of in vitro culture. In one embodiment, the cells are transfected using a minicircle on days 1, 2, 3, 4, 5, 6, 7, 8, or 9 of in vitro culture.

[0113] In one embodiment, cells are transfected using a Sleeping Beauty transposon (e.g., a T2 or T4 Sleeping Beauty transposon) on a day ranging from day 1 to day 12 of in vitro culture. In one embodiment, cells are transduced by electroporation using a Sleeping Beauty transposon system (e.g., a T2 or T4 Sleeping Beauty transposon) on day 2 of in vitro culture. In one embodiment, cells are transduced by electroporation using a Sleeping Beauty transposon system (e.g., a T2 or T4 Sleeping Beauty transposon) on day 5 of in vitro culture. In one embodiment, cells are transduced by electroporation using a Sleeping Beauty transposon system (e.g., a T2 or T4 Sleeping Beauty transposon) on day 8 of in vitro culture. In one embodiment, cells are transduced by electroporation using a Sleeping Beauty transposon (e.g., a T2 or T4 Sleeping Beauty transposon) system on day 11 of in vitro culture. In one embodiment, cells are transduced by electroporation using a Sleeping Beauty transposon (e.g., a T2 or T4 Sleeping Beauty transposon) system on day 14 of in vitro culture.

[0114] In one embodiment, the cells are transfected with a Piggybac transposon on a day ranging from day 1 to day 12 of in vitro culture. In one embodiment, the cells are transduced with a Piggybac transposon by electroporation on day 2 of in vitro culture. In one embodiment, the cells are transduced with a Piggybac transposon by electroporation on day 5 of in vitro culture. In one embodiment, the cells are transduced with a Piggybac transposon by electroporation on day 8 of in vitro culture. In one embodiment, the cells are transduced with a Piggybac transposon by electroporation on day 9 of in vitro culture. In one embodiment, the cells are transduced with a Piggybac transposon on day 11 of in vitro culture. In one embodiment, the cells are transduced with a Piggybac transposon on day 14 of in vitro culture by electroporation.

[0115] In one embodiment, B cells are contacted with a vector comprising a nucleic acid of interest operably linked to a promoter under conditions sufficient to transfect at least a portion of the B cells. In one embodiment, B cells are contacted with a vector comprising a nucleic acid of interest operably linked to a promoter under conditions sufficient to transfect at least 5% of the B cells. In a further embodiment, B cells are contacted with the vector under conditions sufficient to transfect at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even 100% of the B cells. In one specific embodiment, B cells cultured in vitro are transfected as described herein, where the cultured B cells are contacted with a vector described herein under conditions sufficient to transfect at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even 100% of the B cells.

[0116] Viral vectors can be used to transduce memory B cells and / or plasma cells. Examples of viral vectors include, but are not limited to, adenovirus-based vectors, adeno-associated virus (AAV)-based vectors, retrovirus vectors, retrovirus-adenovirus vectors, and vectors derived from herpes simplex virus (HSV), including amplicon vectors, replication-defective HSV, and attenuated HSV (see, for example, Krisky, Gene Ther. 5:1517-30, 1998; Pfeifer, Annu. Rev. Genomics Hum. Genet. 2:177-211, 2001, each of which is incorporated herein by reference in its entirety).

[0117] In one embodiment, the cells are transduced with a viral vector (e.g., a lentiviral vector) on day 1, 2, 3, 4, 5, 6, 7, 8, or 9 of in vitro culture. In a specific embodiment, the cells are transduced with a viral vector on day 5 of in vitro culture. In one embodiment, the viral vector is a lentivirus. In one embodiment, the cells are transduced with a measles virus-pseudotyped lentivirus on day 1 of in vitro culture.

[0118] In one embodiment, B cells are transduced with a retroviral vector using any of a variety of techniques known in the art (see, e.g., Science, April 12, 1996, Vol. 272:263-267; Blood 2007, Vol. 99:2342-2350; Blood 2009, Vol. 113:1422-1431; Blood 2009, October 8, Vol. 114(15):3173-80; Blood. 2003, Vol. 101(6):2167-2174; Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, NY (2009)). Additional descriptions regarding viral transduction of B cells can be found in WO2011 / 085247 and WO2014 / 152832, each of which is incorporated by reference in its entirety.

[0119] For example, PBMCs, B or T lymphocytes, and other B-cell cancer cells, such as B-CLL, from a donor can be isolated and cultured in IMDM medium or RPMI 1640 (GibcoBRL Invitrogen, Auckland, New Zealand), or other suitable medium described herein, either serum-free or supplemented with serum (e.g., 5-10% FCS, human AB serum, and serum replacement) and penicillin / streptomycin, and / or other suitable supplements, such as transferrin and / or insulin. In one embodiment, cells are cultured at 1 x 10 in a 48-well plate. 5 The cells are seeded and the concentrated vector is added at various doses, which can be routinely optimized by those skilled in the art using routine methodology. In one embodiment, B cells are transferred into MS5 cell monolayers in RPMI supplemented with 10% AB serum, 5% FCS, 50 ng / ml rhSCF, 10 ng / ml rhlL-15 and 5 ng / ml rhlL-2, and the medium is periodically refreshed as needed. As will be recognized by those skilled in the art, other suitable media and supplements can be used as desired.

[0120] Certain embodiments relate to the use of retroviral vectors or vectors derived from retroviruses. A "retrovirus" is an enveloped RNA virus that can infect animal cells and utilizes reverse transcriptase to generate DNA copies from its RNA genome during the early stages of infection, which are then typically integrated into the host genome. Examples of retroviral vectors include Moloney murine leukemia virus (MLV)-derived vectors, murine stem cell virus-based retroviral vectors that confer long-term stable expression in target cells such as hematopoietic precursor cells and their differentiated progeny (see, e.g., Hawley et al., PNAS USA 93:10297-10302, 1996; Keller et al., Blood 92:877-887, 1998), hybrid vectors (see, e.g., Choi et al., Stem Cells 19:236-246, 2001), and composite retrovirus-derived vectors such as lentiviral vectors.

[0121] In one embodiment, B cells are contacted with a retroviral vector comprising a nucleic acid of interest operably linked to a promoter under conditions sufficient to transduce at least a portion of the B cells. In one embodiment, B cells are contacted with a retroviral vector comprising a nucleic acid of interest operably linked to a promoter under conditions sufficient to transduce at least 2% of the B cells. In a further embodiment, B cells are contacted with the vector under conditions sufficient to transduce at least 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even 100% of resting B cells. In one specific embodiment, differentiated and activated B cells cultured in vitro as described herein are transduced, where the cultured differentiated / activated B cells are contacted with a vector described herein under conditions sufficient to transduce at least 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even 100% of the differentiated and activated B cells.

[0122] In certain embodiments, prior to transduction, cells are pre-stimulated with Staphylococcus Aureus Cowan (SAC; Calbiochem, San Diego, CA) and / or IL-2 at appropriate concentrations known to those of skill in the art and routinely optimized. Other B cell activators (e.g., PMA) can be used, as known to those of skill in the art and described herein.

[0123] As noted above, certain embodiments utilize lentiviral vectors. The term "lentivirus" refers to a genus of complex retroviruses that can infect both dividing and non-dividing cells. Examples of lentiviruses include HIV (human immunodeficiency virus; including HIV types 1 and 2), visna maedi, caprine arthritis-encephalitis virus, equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV). Lentiviral vectors can be derived from any one or more of these lentiviruses (see, e.g., Evans et al., Hum Gene Ther. 10:1479-1489, 1999; Case et al., PNAS USA 96:2988-2993, 1999; Uchida et al., PNAS USA 95:11939-11944, 1998; Miyoshi et al., Science 283:682-686, 1999; Sutton et al., J Virol 72:5781-5788, 1998; and Frecha et al., Blood. 1 12:4843-52, 2008, each of which is incorporated by reference in its entirety).

[0124] It has been documented that resting T and B cells can be transduced by VSVG-coated LVs that retain most of the HIV accessory proteins (vif, vpr, vpu, and nef) (see, e.g., Frecha et al., 2010, Mol. Therapy 18:1748). In certain embodiments, the retroviral vector comprises certain minimal sequences derived from a lentiviral genome, such as the HIV genome or SIV genome. Lentiviral genomes are typically organized into a 5' long terminal repeat (LTR) region, gag gene, pol gene, env gene, accessory genes (e.g., nef, vif, vpr, vpu, tat, rev), and a 3' LTR region. The viral LTR is divided into three regions designated U3, R (repeat), and U5. The U3 region contains enhancer and promoter elements, the U5 region contains a polyadenylation signal, and the R region separates the U3 and U5 regions. The transcribed sequence of the R region appears at both the 5' and 3' ends of the viral RNA (see, e.g., "RNA Viruses: A Practical Approach," edited by Alan J. Cann, Oxford University Press, 1999, each of which is incorporated by reference in its entirety). Press, 2000); O Narayan, J. Gen. Virology. 70:1617-1639, 1989; Fields et al., Fundamental Virology Raven Press., 1990; Miyoshi et al., J. (See Virol. 72:8150-7, 1998; and U.S. Patent No. 6,013,516.) Lentiviral vectors can include any one or more of these elements of the lentiviral genome to modulate the activity of the vector as desired, or can contain deletions, insertions, substitutions, or mutations in one or more of these elements to, for example, reduce the pathological effects of lentiviral replication or limit the lentiviral vector to a single round of infection.

[0125] Typically, a minimal retroviral vector contains certain 5'LTR and 3'LTR sequences, one or more genes of interest (to be expressed in the target cell), one or more promoters, and cis-acting sequences for packaging RNA. Other regulatory sequences may be included as described herein and known in the art. Viral vectors are typically cloned into a plasmid that can be transfected into a packaging cell line, such as a eukaryotic cell (e.g., 293-HEK), and typically contain sequences useful for replication of the plasmid in bacteria.

[0126] In certain embodiments, the viral vector comprises sequences derived from the 5' and / or 3' LTR of a retrovirus, such as a lentivirus. The LTR sequences can be from any lentivirus from any species. For example, the LTR sequences can be from HIV, SIV, FIV, or BIV. Preferably, the LTR sequences are HIV LTR sequences.

[0127] In certain embodiments, the viral vector comprises the R and U5 sequences from the 5'LTR of a lentivirus and an inactivated or "self-inactivating" 3'LTR from a lentivirus. A "self-inactivating 3'LTR" is a 3'-long terminal repeat (LTR) containing a mutation, substitution, or deletion that prevents the LTR sequence from driving the expression of downstream genes. A copy of the U3 region from the 3'LTR serves as a template for the generation of both LTRs in the integrated provirus. Thus, when a 3'LTR with an inactivating deletion or mutation integrates as the 5'LTR of a provirus, transcription from the 5'LTR is impossible. This eliminates competition between the viral enhancer / promoter and any internal enhancer / promoter. Self-inactivating 3'LTRs are described, for example, in Zufferey et al., J. Virol. 72:9873-9880, 1998; Miyoshi et al., J. Virol. 72:8150-8157, 1998; and Iwakuma et al., J. Virology 261:120-132, 1999, each of which is incorporated by reference in its entirety. Self-inactivating 3'LTRs can be generated by any method known in the art. In certain embodiments, the U3 element of the 3'LTR has a deletion in its enhancer sequence, preferably the TATA box, Spl and / or NF-kappa B site. As a result of the self-inactivating 3'LTR, the provirus integrated into the host cell genome will contain an inactivated 5'LTR.

[0128] The vectors provided herein typically include a gene encoding a protein, e.g., follistatin, that is desired to be expressed in one or more target cells. However, the vectors provided herein also include genes encoding other molecules (e.g., siRNAs) that are desired to be expressed in one or more target cells. In some embodiments, in a viral vector, the gene of interest (e.g., follistatin) is preferably located between the 5'LTR and 3'LTR sequences. Furthermore, in some embodiments, the gene of interest (e.g., follistatin) is preferably in a functional relationship with other genetic elements, e.g., transcriptional regulatory sequences, such as promoters and / or enhancers, that regulate the expression of the gene of interest (e.g., follistatin) in a specific manner once the gene is introduced into the target cells. In certain embodiments, useful transcriptional regulatory sequences are sequences that are highly regulated in terms of activity, both temporally and spatially.

[0129] In certain embodiments, one or more additional genes can be incorporated as a safety measure to allow for selective killing or depletion of transfected target cells within a heterogeneous population, such as within a human patient. In one non-limiting exemplary embodiment, the gene is a thymidine kinase gene (TK), the expression of which sensitizes target cells to the action of the drug ganciclovir. In some embodiments, the additional gene is a cell surface protein tag. In some embodiments, the gene is a suicide gene. In some embodiments, the suicide gene is the caspase-9 suicide gene, which is activated by a dimerizing drug (see, e.g., Tey et al., Biology of Blood and Marrow Transplantation 13:913-924, 2007).

[0130] In certain embodiments, one or more additional genes encoding marker proteins can be placed before or after the primary gene (e.g., the follistatin gene) in a viral or non-viral vector to allow for identification and / or selection of cells expressing the desired protein (e.g., follistatin). Certain embodiments incorporate additional cell surface proteins that can facilitate identification and / or selection of cells expressing the desired protein (e.g., follistatin). Certain embodiments incorporate a fluorescent marker protein, such as green fluorescent protein (GFP) or red fluorescent protein (RFP), along with the primary gene of interest (e.g., the follistatin gene). When one or more additional reporter genes are included, an IRES sequence or 2A element may also be included that separates the primary gene of interest (e.g., the follistatin gene) from the reporter gene and / or any other genes of interest.

[0131] Certain embodiments employ genes encoding one or more selectable markers. Examples include selectable markers effective in eukaryotic or prokaryotic cells, such as genes for drug resistance that encode factors necessary for the survival or growth of transformed host cells grown in selective culture media. Exemplary selectable genes encode proteins that confer resistance to antibiotics or other toxins, such as G418, hygromycin B, puromycin, zeocin, ouabain, blasticidin, ampicillin, neomycin, methotrexate, or tetracycline, or that complement or supplement auxotrophic deficiencies, which may be present on separate plasmids and introduced by cotransfection with a viral vector. In one embodiment, the gene encodes a mutant dihydrofolate reductase (DHFR) that confers resistance to methotrexate. Certain other embodiments may use genes encoding one or more cell surface receptors that can be used to tag and detect or purify transfected cells (e.g., low affinity nerve growth factor receptor (LNGFR)), or other such receptors useful as transduction tagging systems. See, e.g., Lauer et al., Cancer Gene Ther. 2000 March;7(3):430-7.

[0132] Certain viral vectors, such as retroviral vectors, use one or more heterologous promoters, enhancers, or both. In certain embodiments, the U3 sequence from retroviral or lentiviral 5'LTR can be replaced with the promoter or enhancer sequence in the viral construct. Certain embodiments use an "internal" promoter / enhancer located between the 5'LTR and 3'LTR sequences of the viral vector, operably linked to the gene of interest (e.g., the follistatin gene).

[0133] "Functional relationship" and "operably linked" mean, without limitation, that the gene (e.g., the follistatin gene) is in the correct location and orientation with respect to the promoter and / or enhancer such that, when the promoter and / or enhancer is contacted with the appropriate regulatory molecules, expression of the gene (e.g., the follistatin gene) will be affected. Any enhancer / promoter combination can be used that modulates (e.g., increases, decreases) expression of the viral RNA genome in the packaging cell line, modulates expression of the selected gene of interest in the infected target cells, or both.

[0134] A promoter is an expression control element formed by a DNA sequence that allows polymerase binding and transcription to occur. A promoter is a non-translated sequence located upstream (5') of the start codon of a selected gene of interest (typically within about 100-1000 bp) that controls the transcription and translation of an operably linked coding polynucleotide sequence. Promoters can be inducible or constitutive. An inducible promoter initiates increased levels of transcription from DNA under its control in response to some change in culture conditions, such as a change in temperature. Promoters can be unidirectional or bidirectional. A bidirectional promoter can be used to simultaneously express two genes, e.g., a gene of interest such as follistatin and a selectable marker. Alternatively, a bidirectional promoter construct can be used, containing two promoters in opposite orientations in the same vector, each controlling the expression of a different gene.

[0135] A variety of promoters are known in the art, as well as the method for operably linking promoters to polynucleotide coding sequences.Both native promoter sequences and many heterologous promoters can be used to direct the expression of selected target genes.Some embodiments generally use heterologous promoters, because they generally allow better transcription and higher yield of desired protein compared with native promoters.

[0136] Certain embodiments can use heterologous viral promoters. Examples of such promoters include promoters derived from the genomes of viruses such as polyomavirus, fowlpox virus, adenovirus, bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and simian virus 40 (SV40). Certain embodiments can use heterologous mammalian promoters, such as actin promoters, immunoglobulin promoters, heat shock promoters, or promoters associated with the native sequence of a gene of interest (e.g., the follistatin gene). Typically, the promoter is compatible with the target cell, such as activated B lymphocytes, plasma B cells, memory B cells, or other lymphoid target cells.

[0137] Certain embodiments may use one or more RNA polymerase II and III promoters. Suitable selection of RNA polymerase III promoters can be found, for example, in Paule and White, Nucleic Acids Research, Vol. 28, pp. 1283-1298, 2000, incorporated herein by reference in its entirety. RNA polymerase II and III promoters also include any synthetic or engineered DNA fragment capable of directing RNA polymerase II or III, respectively, to transcribe its downstream RNA coding sequence. Furthermore, the RNA polymerase II or III (Pol II or III) promoter(s) used as part of the viral vector can be inducible. Any suitable inducible Pol II or III promoter can be used with the methods described herein. Exemplary Pol II or III promoters include tetracycline-responsive promoters, as provided in Ohkawa and Taira, Human Gene Therapy, Vol. 11, pp. 577-585, 2000; and Meissner et al., Nucleic Acids Research, Vol. 29, pp. 1672-1682, 2001, each of which is incorporated by reference in its entirety.

[0138] Non-limiting examples of constitutive promoters that can be used include the promoter for ubiquitin, the CMV promoter (see, e.g., Karasuyama et al., J. Exp. Med. 169:13, 1989), the β-actin promoter (see, e.g., Gunning et al., PNAS USA 84:4831-4835, 1987), the elongation factor-1 alpha (EF-1 alpha) promoter, the CAG promoter, and the pgk promoter (see, e.g., Adra et al., Gene 60:65-74, 1987; Singer-Sam et al., Gene 32:409-417, 1984; and Dobson et al., Nucleic Acids Res. 10:2635-2637, 1982). Non-limiting examples of tissue-specific promoters include the lck promoter (see, e.g., Garvin et al., Mol. Cell Biol. 8:3058-3064, 1988; and Takadera et al., Mol. Cell Biol. 9:2173-2180, 1989), the myogenin promoter (Yee et al., Genes and Development 7:1277-1289, 1993), and the thyl promoter (see, e.g., Gundersen et al., Gene 1 13:207-214, 1992).

[0139] Additional examples of promoters include the ubiquitin-C promoter, the human μ heavy chain promoter or Ig heavy chain promoter (e.g., MH), and the human kappa light chain promoter or Ig light chain promoter (e.g., EEK), which are functional in B lymphocytes. The MH promoter contains a human μ heavy chain promoter preceded by an iEμ enhancer flanked by matrix-associated regions, and the EEK promoter contains a kappa light chain promoter preceded by an intronic enhancer (iEκ), a matrix-associated region, and a 3′ enhancer (3Eκ) (see, e.g., Luo et al., Blood. 1 13:1422-1431, 2009, and U.S. Patent Application Publication No. 2010 / 0203630). Therefore, certain embodiments may use one or more of these promoter or enhancer elements.

[0140] In one embodiment, one promoter drives expression of a selectable marker and a second promoter drives expression of a gene of interest (e.g., the follistatin gene). For example, in one embodiment, the EF-1 alpha promoter drives production of a selectable marker (e.g., DHFR) and a miniature CAG promoter (see, e.g., Fan et al., Human Gene Therapy 10:2273-2285, 1999) drives expression of the gene of interest (e.g., follistatin).

[0141] As noted above, certain embodiments use enhancer elements, such as internal enhancers, to increase expression of a gene of interest. Enhancers are cis-acting elements of DNA, typically about 10-300 bp in length, that act on a promoter to increase its transcription. Enhancer sequences can be derived from mammalian genes (e.g., globin, elastase, albumin, α-fetoprotein, insulin), such as the ιεμ enhancer, the ιεκ intronic enhancer, and the 3′εκ enhancer. Enhancers derived from eukaryotic viruses are also included, including the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Enhancers can be spliced ​​into the vector at positions 5′ or 3′ relative to the antigen-specific polynucleotide sequence, but are preferably located 5′ from the promoter. One of skill in the art would be able to select an appropriate enhancer based on the desired expression pattern.

[0142] In certain embodiments, a promoter is selected to allow inducible expression of a gene of interest (e.g., the follistatin gene). Numerous systems for inducible expression are known in the art, including the tetracycline-responsive system and the lac operator-repressor system. It is also contemplated that a combination of promoters can be used to obtain the desired expression of a gene of interest (e.g., the follistatin gene). One skilled in the art would be able to select a promoter based on the desired expression pattern of the gene in the organism and / or target cell of interest.

[0143] Certain viral vectors contain cis-acting packaging sequences to facilitate the incorporation of genomic viral RNA into viral particles. Examples include psi-sequences. Such cis-acting sequences are known in the art. In certain embodiments, the viral vectors described herein can express two or more genes, which can be achieved by incorporating an internal promoter operably linked to each of the separate genes beyond the first gene, for example, by incorporating elements that facilitate simultaneous expression, such as an internal ribosomal entry sequence (IRES) element (U.S. Pat. No. 4,937,190, incorporated herein by reference) or a 2A element, or both. Simply by way of illustration, an IRES or 2A element can be used when a single vector contains sequences encoding each chain of an immunoglobulin molecule with the desired specificity. For example, a first coding region (encoding either the heavy or light chain) can be located immediately downstream from the promoter, a second coding region (encoding the other chain) can be located downstream from the first coding region, and an IRES or 2A element is located between the first and second coding regions, preferably immediately preceding the second coding region. In other embodiments, an IRES or 2A element is used to co-express unrelated genes, such as reporter genes, selectable markers, cell surface proteins, or genes that enhance immune function. Examples of IRES sequences that can be used include, without limitation, the IRES elements of encephalomyelitis virus (EMCV), foot-and-mouth disease virus (FMDV), Theiler's murine encephalomyelitis virus (TMEV), human rhinovirus (HRV), coxsackievirus (CSV), poliovirus (POLIO), hepatitis A virus (HAV), hepatitis C virus (HCV), and pestiviruses (e.g., hog cholera virus (HOCV) and bovine viral diarrhea virus (BVDV)) (see, e.g., Le et al., Virus Genes, 2002, 144:1457-1462, each of which is incorporated by reference in its entirety). 12:135-147, 1996; and Le et al., Nuc. Acids Res. 25:362-369, 1997.) An example of a 2A element is the F2A sequence from foot-and-mouth disease virus.

[0144] In certain embodiments, the vectors provided herein also contain additional genetic elements to achieve a desired result. For example, certain viral vectors can include a signal that facilitates nuclear entry of the viral genome in target cells, such as the HIV-1 flap signal. As yet another example, certain viral vectors can include elements that facilitate characterization of proviral integration sites in target cells, such as a tRNA amber suppressor sequence. Certain viral vectors can contain one or more genetic elements designed to enhance expression of a gene of interest (e.g., the follistatin gene). For example, a woodchuck hepatitis virus responsive element (WRE) can be placed in the construct (see, e.g., Zufferey et al., J. Virol. 74:3668-3681, 1999; and Deglon et al., Hum. Gene Ther. 11:179-190, 2000, each of which is incorporated by reference in its entirety). As another example, a chicken β-globin insulator can be included in the construct. This element has been shown to reduce the probability of integrated DNA silencing in target cells due to methylation and heterochromatinization effects. Additionally, the insulator can shield internal enhancers, promoters, and exogenous genes from positive or negative positional effects from surrounding DNA at the integration site on the chromosome. Certain embodiments utilize each of these genetic elements. In another embodiment, the viral vectors provided herein can also contain a ubiquitous chromatin opening element (UCOE) to increase expression (see, e.g., Zhang F et al., Molecular Therapy: The journal of the American Society of Gene Therapy, September 2010; 18(9):1640-9).

[0145] In certain embodiments, the viral vectors provided herein (e.g., retroviruses, lentiviruses) are "pseudotyped" with one or more selected viral glycoproteins or envelope proteins primarily for targeting selected cell types. Pseudotyping generally refers to the incorporation of one or more heterologous viral glycoproteins onto cell surface viral particles, which often enables the viral particles to infect selected cells that are different from their normal target cells. The "heterologous" element is derived from a virus other than the virus from which the RNA genome of the viral vector is derived. Typically, the glycoprotein coding region of the viral vector is genetically altered, such as by deletion to prevent expression of its own glycoprotein. Simply by way of illustration, the envelope glycoproteins gp41 and / or gp120 from HIV-derived lentiviral vectors are typically deleted before pseudotyping with heterologous viral glycoproteins.

[0146] In certain embodiments, the viral vector is pseudotyped with a heterologous viral glycoprotein that targets B lymphocytes. In certain embodiments, the viral glycoprotein allows for selective infection or transduction of resting or quiescent B lymphocytes. In certain embodiments, the viral glycoprotein allows for selective infection of B lymphocytes, plasma cells, plasmablasts, and activated B cells. In certain embodiments, the viral glycoprotein allows for infection or transduction of resting B lymphocytes, plasmablasts, plasma cells, and activated B cells. In certain embodiments, the viral glycoprotein allows for infection of B-cell chronic lymphocytic leukemia cells. In one embodiment, the viral vector is pseudotyped with VSV-G. In another embodiment, the heterologous viral glycoprotein is derived from the glycoprotein of a measles virus, such as Edmonton measles virus. Certain embodiments pseudotype the measles virus glycoprotein hemagglutinin (H), the fusion protein (F), or both (see, e.g., Frecha et al., Blood. 1 12:4843-52, 2008; and Frecha et al., Blood. 1 14:3173-80, 2009, each of which is incorporated by reference in its entirety). In one embodiment, the viral vector is pseudotyped with gibbon leukemia virus (GALV). In one embodiment, the viral vector is pseudotyped with feline endogenous retrovirus (RD114). In one embodiment, the viral vector is pseudotyped with baboon endogenous retrovirus (BaEV). In one embodiment, the viral vector is pseudotyped with murine leukemia virus (MLV). In one embodiment, the viral vector is pseudotyped with gibbon leukemia virus (GALV). In further embodiments, the viral vector contains a buried antibody binding domain, such as one or more variable regions (e.g., heavy and light chain variable regions), that function to target the vector to a specific cell type.

[0147] Production of viral vectors can be accomplished using any suitable genetic engineering technique known in the art, including, but not limited to, standard techniques of restriction endonuclease digestion, ligation, transformation, plasmid purification, PCR amplification, and DNA sequencing, as described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY (1989)), Coffin et al. (Retroviruses. Cold Spring Harbor Laboratory Press, NY (1997)), and "RNA Viruses: A Practical Approach" (Alan J. Cann, ed., Oxford University Press (2000)).

[0148] Any of a variety of methods known in the art can be used to produce suitable retroviral particles whose genomes contain RNA copies of the viral vector. In one method, the viral vector can be introduced into a packaging cell line that packages the viral genomic RNA based on the viral vector into viral particles with the desired target cell specificity. The packaging cell line typically provides, in trans, the viral proteins required for packaging the viral genomic RNA into viral particles and for infection of target cells, including the structural gag protein, the enzymatic pol protein, and envelope glycoproteins.

[0149] In certain embodiments, the packaging cell line stably expresses certain necessary or desired viral proteins (e.g., gag, pol) (see, e.g., U.S. Patent No. 6,218,181, incorporated herein by reference). In certain embodiments, the packaging cell line is transiently transfected with a plasmid encoding certain necessary or desired viral proteins (e.g., gag, pol, glycoproteins), including the measles virus glycoprotein sequences described herein. In an exemplary embodiment, the packaging cell line stably expresses gag and pol sequences, and the cell line is then transfected with a plasmid encoding the viral vector and a plasmid encoding the glycoprotein. After introduction of the desired plasmid, the viral particles are harvested and processed accordingly, such as by ultracentrifugation, to achieve a concentrated stock of viral particles. Exemplary packaging cell lines include 293 (ATCC CCL X), HeLa (ATCC CCL 2), D17 (ATCC CCL 183), MDCK (ATCC CCL 34), BHK (ATCC CCL-10), and Cf2Th (ATCC CRL 1430) cell lines.

[0150] therapeutic agent As used herein, "gene of interest" or "gene" or "nucleic acid of interest" refers to a transgene to be expressed in a target transfected cell. In certain embodiments, the gene is a follistatin gene. While the term "gene" can be used, this term does not imply that the gene is found in genomic DNA and is used interchangeably with the term "nucleic acid." Generally, a nucleic acid of interest provides a nucleic acid suitable for encoding a therapeutic agent (e.g., follistatin) and can comprise cDNA or DNA, and may or may not contain introns, but generally does not contain introns. As noted elsewhere, the nucleic acid of interest is operably linked to an expression control sequence for effectively expressing the protein of interest in the target cell. In certain embodiments, the vectors described herein can contain one or more genes of interest, for example, two, three, four, five, or more genes of interest, such as immunoglobulin heavy and light chains, which can be organized using the internal promoters described herein.

[0151] Reference to "polynucleotide" or "nucleic acid" herein designates mRNA, RNA, cRNA, cDNA, or DNA. This term typically refers to a polymeric form of nucleotides at least 10 bases in length, which are ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide. This term includes single- and double-stranded forms of DNA and RNA. The nucleic acid or gene of interest can be any nucleic acid that encodes a protein of interest.

[0152] In some embodiments, any one of the embodiments disclosed herein can utilize a gene of interest that is a follistatin protein. The follistatin protein, in some embodiments, can be any one of the follistatin proteins set forth in SEQ ID NOS: 1-4. Thus, in some embodiments, a therapeutic agent delivered to genetically modified B cells as described herein can be a follistatin protein.

[0153] Follistatin In various aspects, the present disclosure relates to B cells engineered to express follistatin (e.g., one or more follistatin peptides). As used herein, the term "follistatin" refers to the follistatin (FST) protein family and follistatin-related proteins from any species. Follistatin is an autocrine glycoprotein expressed in almost all tissues of higher animals. Follistatin was first isolated from follicular fluid and identified as a protein fraction that inhibits the secretion of follicle-stimulating hormone (FSH) from the anterior pituitary gland, and was therefore designated FSH-suppressing protein (FSP). Its primary function was then determined to be the binding and neutralization of members of the TGF-β superfamily, including, for example, activin, a paracrine hormone that enhances FSH secretion in the anterior pituitary gland.

[0154] In some embodiments, the terms "follistatin polypeptide" or "follistatin" are used to refer to polypeptides including any naturally occurring polypeptide of the follistatin family and any variants thereof (mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity, including, for example, ligand binding (e.g., myostatin, GDF-11, activin A, activin B) or heparin binding. For example, in some embodiments, a follistatin polypeptide may include a polypeptide comprising an amino acid sequence derived from the sequence of any known follistatin having a sequence at least about 80% identical to the sequence of a follistatin polypeptide, preferably at least 85%, 90%, 95%, 97%, 99% or higher percent identity.

[0155] Follistatin is a single-chain polypeptide with a molecular weight ranging from 31 to 49 kDa due to alternative splicing of the mRNA and variable glycosylation of the protein. The human gene encoding follistatin (FST) contains six exons spanning 5,329 bp on chromosome 5q11.2, generating two major transcripts: transcript variant FST344 (1,122 bp) and transcript FST317 (1,386 bp). Exon 1 in FST encodes the follistatin signal peptide, exon 2 encodes the follistatin N-terminal domain, and exons 3 to 5 each encode a follistatin module. Alternative splicing allows the utilization of either exon 6A (encoding the acidic region in FST344) or exon 6B (containing two bases of the stop codon in FST317) (Shimasaki, S. et al., 1988).

[0156] These alternatively spliced ​​mRNAs (FST344 and FST317) produce two follistatin proteins of 315 amino acids (i.e., FST315) and 288 amino acids (i.e., FST288), respectively, after removal of the 29-amino acid signal peptide. Follistatin 315 can be further proteolytically cleaved to follistatin 303 (FST303). Amino acid sequence analysis revealed that the native human follistatin polypeptide contains five domains (from the N-terminus): a signal sequence peptide (amino acids 1-29 of SEQ ID NO:1), an N-terminal domain (FSN) (amino acids 30-94 of SEQ ID NO:1), follistatin domain I (FSDI) (amino acids 95-164 of SEQ ID NO:1), follistatin domain II (FSDII) (amino acids 168-239 of SEQ ID NO:1), and follistatin domain III (FSDIII) (amino acids 245-316 of SEQ ID NO:1). See PNAS, USA, 1988, Vol. 85, No. 12, pp. 4218-4222.

[0157] Human follistatin-288 (FST288) precursor (i.e., FST317) has the following amino acid sequence, with the signal peptide shown in bold, the N-terminal domain (FSN) single underlined, and follistatin domains I-III (FSI, FSII, FSIII) double underlined: [ka]

[0158] The processed (mature) human follistatin variant (FST288) has the following amino acid sequence, with the N-terminal domain single-underlined and follistatin domains I-III double-underlined: It is further recognized that either the first amino acid G or N before the first cysteine ​​can be removed or intentionally eliminated by processing, regardless of the consequences, and further includes polypeptides including such slightly smaller polypeptides. [ka]

[0159] Human follistatin-315 (FST315) precursor (i.e., FST344) has the following amino acid sequence, with the signal peptide in bold, the N-terminal domain (FSN) single underlined, and follistatin domains I-III (FSI, FSII, FSIII) double underlined (NCBI accession number AAH04107.1; 344 amino acids): [ka]

[0160] Processed (mature) human FST315 has the following amino acid sequence, with the N-terminal domain single-underlined and follistatin domains I-III double-underlined: It is further recognized that either the first amino acid G or N before the first cysteine ​​can be removed or intentionally eliminated by processing, regardless of the consequences, and further includes polypeptides including such slightly smaller polypeptides. [ka]

[0161] Follistatin polypeptides of the present disclosure may include any naturally occurring domain of the follistatin protein and variants thereof (e.g., mutants, fragments, and peptidomimetic forms) that retain useful activity. For example, it is well known that FST315 and FST288 have high affinity for both activins (activin A and activin B) and myostatin (and the closely related GDF11), and follistatin domains (e.g., FSN and FSDI-III) are thought to be involved in binding such TGF-β ligands. However, it is believed that each of these three domains may have different affinities for such TGF-β ligands. For example, one study demonstrated that a polypeptide construct containing a single N-terminal domain (FSN) and two FSDI domains in tandem retained high affinity for myostatin and little or no affinity for activin when introduced into mice by genetic expression, promoting whole-body muscle growth (Nakatani et al., The FASEB Journal, Vol. 22477-487 (2008)).

[0162] Thus, the present disclosure encompasses, in part, variant follistatin proteins that demonstrate selective binding and / or inhibition of a given TGF-β ligand to a naturally occurring FST protein (e.g., having significantly reduced affinity for activin while maintaining high affinity for myostatin).

[0163] Thus, this disclosure provides polynucleotides (isolated, purified, or pure polynucleotides) encoding a therapeutic agent of the present disclosure (e.g., follistatin) for genetically modifying B cells, vectors (including cloning vectors and expression vectors) containing such polynucleotides, and cells (e.g., host cells) transformed or transfected with a polynucleotide or vector according to the present disclosure. In certain embodiments, any one of the embodiments disclosed in this disclosure may utilize a follistatin (e.g., for expression in B cells) selected from the follistatin polypeptides of SEQ ID NOs: 1-4. In certain embodiments, any one of the embodiments disclosed in this disclosure may utilize a follistatin (e.g., for expression in B cells) that is a human follistatin FST344 splice site variant. In certain embodiments, polynucleotides (DNA or RNA) encoding a protein of interest (e.g., follistatin) of the present disclosure are contemplated. Expression cassettes encoding proteins of interest are also contemplated herein.

[0164] The present disclosure also relates to vectors comprising the polynucleotides of the present disclosure, particularly recombinant expression constructs. In one embodiment, the present disclosure contemplates a vector comprising a polynucleotide encoding a protein of the present disclosure (e.g., follistatin) together with other polynucleotide sequences that cause or facilitate the transcription, translation, and processing of such protein-coding sequences. Cloning and expression vectors suitable for use with prokaryotic and eukaryotic hosts are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, NY (1989). Exemplary cloning / expression vectors include cloning vectors, shuttle vectors, and expression constructs that can be based on plasmids, phagemids, phasmids, cosmids, viruses, artificial chromosomes, or any nucleic acid vehicle known in the art that is suitable for amplifying, transferring, and / or expressing a polynucleotide contained therein.

[0165] As used herein, unless otherwise specified with respect to viral vectors, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked thereto. Exemplary vectors include plasmids, minicircles, transposons (e.g., Sleeping Beauty transposons), yeast artificial chromosomes, self-replicating RNA, and viral genomes. Certain vectors can replicate autonomously in host cells, while other vectors can integrate into the genome of the host cell and thereby replicate together with the host genome. In addition, certain vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors") that contain nucleic acid sequences operably linked to expression control sequences (thus allowing the expression of such sequences to be directed). In certain embodiments, the expression construct is derived from a plasmid vector. Illustrative constructs include the modified pNASS vector (Clontech, Palo Alto, CA), which has a nucleic acid sequence encoding an ampicillin resistance gene, a polyadenylation signal, and a T7 promoter site; pDEF38 and pNEF38 (CMC ICOS Biologics, Inc.), which have the CHEF1 promoter; and pD18 (Lonza), which has the CMV promoter. Other suitable mammalian expression vectors are well known (see, e.g., Ausubel et al., 1995; Sambrook et al., supra; see also, e.g., catalogs from Invitrogen, San Diego, CA; Novagen, Madison, WI; and Pharmacia, Piscataway, NJ).

[0166] Useful constructs can be prepared that contain a dihydrofolate reductase (DHFR) coding sequence under regulatory control suitable for promoting enhanced production levels of the fusion protein, resulting from gene amplification following application of an appropriate selection agent (e.g., methotrexate). In one embodiment, the use of a bifunctional transposon encoding a therapeutic gene (e.g., FST) along with a drug-resistant DHFR gene, combined with incubation in methotrexate (MTX) to enrich for successfully translocated B cells, generates a more potent product.

[0167] Generally, recombinant expression vectors will contain an origin of replication and a selectable marker, as described above, to enable transformation of host cells, as well as a promoter derived from a highly expressed gene to direct transcription of downstream structural sequences. A vector operably linked to a polynucleotide of the present disclosure results in a cloning or expression construct. Exemplary cloning / expression constructs contain at least one expression control element, e.g., a promoter, operably linked to a polynucleotide of the present disclosure. Additional expression control elements, such as enhancers, factor-specific binding sites, terminators, and ribosome binding sites, are also contemplated in the vectors and cloning / expression constructs of the present disclosure. The heterologous structural sequence of the polynucleotide of the present disclosure is assembled in appropriate phase with translation initiation and termination sequences. Thus, for example, the encoding nucleic acids provided herein can be included in any one of a variety of expression vector constructs (e.g., minicircles) as recombinant expression constructs for expressing such proteins in host cells.

[0168] The appropriate DNA sequence(s) can be inserted into a vector, for example, by a variety of procedures. Generally, the DNA sequence is inserted into an appropriate restriction endonuclease cleavage site(s) by procedures known in the art. Standard techniques for cloning, DNA isolation, amplification and purification, as well as various separation techniques for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases, etc., are contemplated. Numerous standard techniques are described, for example, in Ausubel et al. (Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., Boston, MA, 1993); Sambrook et al. (Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory, Plainview, NY, 1989); Maniatis et al. (Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, NY, 1999); Spring Harbor Laboratory, Plainview, NY, 1982); Glover (ed.) (DNA Cloning, Vols. I and II, IRL Press, Oxford, UK, 1985); Hames and Higgins (eds.) (Nucleic Acid Hybridization, IRL Press, Oxford, UK, 1985); and others.

[0169] The DNA sequence in the expression vector is operably linked to at least one appropriate expression control sequence (e.g., a constitutive promoter or a regulatable promoter) to direct mRNA synthesis. Representative examples of such expression control sequences include promoters of eukaryotic cells or their viruses, as described above. Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors, kanamycin vectors, or other vectors with selectable markers. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retroviruses, and mouse metallothionein-1. Selection of appropriate vectors and promoters is well within the skill of those in the art. The preparation of certain particularly preferred recombinant expression constructs containing at least one promoter or regulatable promoter operably linked to a nucleic acid encoding a protein or polypeptide of the present disclosure is described herein.

[0170] Variants of the polynucleotides of the present disclosure are also contemplated. Variant polynucleotides are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, and preferably 95%, 96%, 97%, 98%, 99%, or 99.9% identical to one of the polynucleotides of defined sequences described herein, or hybridize to one of the polynucleotides of defined sequences under stringent hybridization conditions of 0.015 M sodium chloride, 0.0015 M sodium citrate at about 65-68°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at about 42°C. Polynucleotide variants retain the ability to encode functional binding domains or fusion proteins thereof as described herein.

[0171] The term "stringent" is used to refer to conditions generally understood as stringent in the art. Hybridization stringency is primarily determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at about 65-68°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at about 42°C (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). More stringent conditions (such as higher temperature, lower ionic strength, or higher concentration of formamide or other denaturing agents) can also be used; however, the rate of hybridization will be affected. Where deoxyoligonucleotide hybridization is concerned, additional exemplary stringent hybridization conditions include washing in 6×SSC, 0.05% sodium pyrophosphate at 37° C. (for 14-base oligonucleotides), 48° C. (for 17-base oligonucleotides), 55° C. (for 20-base oligonucleotides), and 60° C. (for 23-base oligonucleotides).

[0172] A further aspect of the present disclosure provides host cells transformed or transfected with or otherwise containing any of the polynucleotides or vectors / expression constructs of the present disclosure (e.g., follistatin polynucleotides or vectors / expression constructs). The polynucleotides or cloning / expression constructs of the present disclosure are introduced into suitable cells using any method known in the art (e.g., any of the methods disclosed herein), including transformation, transfection, and transduction. Host cells include, for example, cells of a subject undergoing ex vivo cell therapy, including ex vivo gene therapy. Eukaryotic host cells contemplated as embodiments of the present disclosure for carrying a polynucleotide, vector, or protein of the present disclosure include, in addition to the subject's own cells (e.g., a human patient's own cells), VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines (including modified CHO cells that can modify the glycosylation pattern of the expressed multivalent binding molecule; see U.S. Patent Application Publication No. 2003 / 0115614), COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562, HEK293 cells, HepG2 cells, N cells, 3T3 cells, Spodoptera frugiperda cells (e.g., Sf9 cells), Saccharomyces cerevisiae cells, and any other eukaryotic cells known in the art to be useful for the expression and, optionally, isolation of proteins or peptides of the present disclosure. Prokaryotic cells are also contemplated, including Escherichia coli, Bacillus subtilis, Salmonella typhimurium, Streptomyces, or any prokaryotic cell known in the art to be suitable for expression and, if necessary, isolation of the proteins or peptides of the present disclosure. It is contemplated that techniques known in the art for extracting proteins from inclusion bodies, in particular, can be used to isolate proteins or peptides from prokaryotic cells. The selection of an appropriate host is within the skill of one of ordinary skill in the art, given the teachings herein. Host cells that glycosylate the fusion proteins of the present disclosure are contemplated.

[0173] The term "recombinant host cell" (or simply "host cell") refers to a cell that contains a recombinant expression vector. It should be understood that the term is intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in successive generations, either due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" herein. Recombinant host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying particular genes. Culture conditions, such as temperature, pH, and the like, for the particular host cell selected for expression will be readily apparent to those skilled in the art. Various mammalian cell culture systems can also be used to express recombinant proteins. Examples of mammalian expression systems include the COS-7 line of monkey kidney fibroblasts described by Gluzman (1981) Cell 23:175, as well as other cell lines capable of expressing compatible vectors, such as C127, 3T3, CHO, HeLa, and BHK cell lines. Mammalian expression vectors will also contain an origin of replication, a suitable promoter and, optionally, an enhancer, as well as any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5'-flanking nontranscribed sequences, such as those described herein in consideration of the preparation of multivalent binding protein expression constructs. DNA sequences derived from the SV40 splice and polyadenylation sites can be used to provide the required nontranscribed genetic elements. Introduction of the construct into host cells can be effected by a variety of methods familiar to those skilled in the art, including calcium phosphate transfection, DEAE-dextran-mediated transfection, or electroporation (Davis et al. (1986) Basic Methods in Molecular Biology).

[0174] Cells and compositions In one embodiment, the engineered B cells described herein have been activated / differentiated in vitro and transfected to express a therapeutic agent described herein (e.g., follistatin). In one embodiment, the engineered B cells described herein have been activated / differentiated in vitro and engineered (e.g., using a targeted transgene integration approach, such as zinc finger nucleases, TALENs, meganucleases, or CRISPR / CAS9-mediated transgene integration) to express a therapeutic agent described herein (e.g., follistatin). In one embodiment, the composition comprises B cells that have been differentiated into plasma B cells and transfected or otherwise engineered to express one or more proteins of interest (e.g., follistatin). Target cell populations, such as the transfected or otherwise engineered activated B cell populations of the present disclosure, can be administered alone or as a pharmaceutical composition in combination with other components, such as diluents and / or cytokines or cell populations.

[0175] In one embodiment, modified B cells engineered to express one or more proteins of interest (e.g., follistatin) are harvested from culture following in vitro activation / differentiation at a time when the modified B cells have optimal migratory capacity for a particular chemoattractant. In some embodiments, optimal migratory capacity may occur on day 7, 8, or 9 of B cell culture. In some embodiments, optimal migratory capacity may occur on day 5, 6, or 7 of B cell culture following transfection or manipulation. In some embodiments, optimal migratory capacity may occur on day 8 of B cell culture following transfection or manipulation, or after day 8 of culture following transfection or manipulation (e.g., on or after day 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or later). In some embodiments, optimal migratory capacity may occur before day 10 of B cell culture. In some embodiments, optimal migratory capacity may occur before day 8 of B cell culture following transfection or manipulation. In some embodiments, optimal migratory capacity may occur on day 6 or 7 of B cell culture. In some embodiments, optimal migratory capacity may occur on day 4 or 5 of B cell culture after transfection or manipulation. In some embodiments, optimal migratory capacity may occur before day 9 of B cell culture. In some embodiments, optimal migratory capacity may occur before day 7 of B cell culture after transfection or manipulation. In some embodiments, optimal migratory capacity is optimal for engineered B cell homing to CXCL12. In some embodiments, optimal migratory capacity is optimal for engineered B cell homing to the bone marrow of a subject receiving one or more administrations of the engineered B cells. In some embodiments, B cells are harvested for administration to the subject at about day 7 to about day 9 in culture, at optimal migratory capacity to CXCL12 and / or the subject's bone marrow. In some embodiments, B cells are harvested for administration to a subject at about day 5 to about day 7 in culture after transfection or manipulation, at optimal CXCL12 and / or migration capacity to the subject's bone marrow.In some embodiments, B cells are collected for administration to a subject at about day 10 in culture, at optimal CXCL12 and / or migratory capacity to the subject's bone marrow. In some embodiments, B cells are collected for administration to a subject at about day 8 in culture after transfection or manipulation, at optimal CXCL12 and / or migratory capacity to the subject's bone marrow. In some embodiments, the optimal migratory capacity is optimal for engineered B cell homing to CXCL13. In some embodiments, the optimal migratory capacity is optimal for engineered B cell homing to a site of inflammation in a subject receiving one or more administrations of the engineered B cells. In some embodiments, B cells are collected for administration to a subject at about day 6 or about day 7 in culture, at optimal CXCL13 and / or migratory capacity to a site of inflammation in the subject. In some embodiments, B cells are collected for administration to a subject at about day 4 or about day 5 in culture after transfection or manipulation, at optimal CXCL13 and / or migration capacity to a site of inflammation in the subject. In some embodiments, B cells are collected for administration to a subject before about day 10 in culture, at optimal CXCL13 and / or migration capacity to a site of inflammation. In some embodiments, B cells are collected for administration to a subject before about day 8 in culture after transfection or manipulation, at optimal CXCL13 and / or migration capacity to a site of inflammation.

[0176] In some embodiments, the optimal migratory capacity is optimal for engineered B cell homing to both CXCL12 and CXCL13. In some embodiments, B cells are harvested on day 7 of B cell culture with optimal migratory capacity for homing to both CXCL12 and CXCL13. In some embodiments, B cells are harvested on day 5 of B cell culture after transfection or manipulation with optimal migratory capacity for homing to both CXCL12 and CXCL13.

[0177] In some embodiments, engineered B cells are collected when at least about 20% of B cells migrate toward a particular chemoattractant in a chemotaxis assay. For example, and not by way of limitation, engineered B cells (e.g., producing FST) can be collected when at least about 20% of B cells migrate toward CXCL12 in a chemotaxis assay. Alternatively, in another non-limiting example, engineered B cells (e.g., producing FST) can be collected when at least about 20% of B cells migrate toward CXCL13 in a chemotaxis assay. Furthermore, engineered B cells (e.g., producing FST) can be collected when at least about 30% of B cells migrate toward a particular chemoattractant (e.g., CXCL12 or CXCL13) in a chemotaxis assay, or when at least about 40%, 45%, 50%, 55%, 60%, 65%, or at least about 70% of B cells migrate toward a particular chemoattractant (e.g., CXCL12 or CXCL13) in a chemotaxis assay. Furthermore, engineered B cells (e.g., producing FST) can be collected when more than 70% of B cells migrate in a chemotaxis assay. Such chemotaxis assays are known in the art.

[0178] Briefly, the cell compositions of the present disclosure can comprise a differentiated and activated B cell population that has been transfected and expresses a therapeutic agent (e.g., follistatin) described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include a buffer such as neutral buffered saline, phosphate buffered saline, lactated Ringer's solution, or the like; a carbohydrate such as glucose, mannose, sucrose, or dextran, mannitol; a protein; a polypeptide; or an amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The compositions of the present disclosure are preferably formulated for intravenous or subcutaneous administration.

[0179] In one embodiment, the cell composition is assessed for purity prior to administration. In another embodiment, the cell composition is tested for robustness of therapeutic agent production. In one embodiment, the cell composition is tested for sterility. In another embodiment, the cell composition is screened to ensure compatibility with the recipient subject.

[0180] In one embodiment, the engineered B cell population is assessed for polyclonality prior to administration to a subject. In some embodiments, ensuring the polyclonality of the final cell product is an important safety parameter. Specifically, the emergence of a dominant clone may be considered a potential contributor to in vivo tumorigenesis or autoimmune disease. Polyclonality can be assessed by any means known in the art or described herein. For example, in some embodiments, polyclonality is assessed by sequencing (e.g., deep sequencing) the B cell receptors expressed in the engineered B cell population. Because B cell receptors change during B cell development and become unique among B cells, this method allows for quantification of how many cells share the same B cell receptor sequence (meaning they are clonal). Thus, in some embodiments, the more B cells in an engineered B cell population express the same B cell receptor sequence, the higher the clonality of the population and, therefore, the less safe the population is for administration to a subject. Conversely, in some embodiments, the fewer B cells in an engineered B cell population that express the same B cell receptor sequence, the less clonality (i.e., the more polyclonal) the population will be, and therefore the safer the population will be for administration to a subject.

[0181] In some embodiments, the engineered B cells are administered to a subject after being determined to be sufficiently polyclonal. For example, the engineered B cells can be administered to a subject after it has been determined that a particular B cell clone in the final population does not constitute more than about 0.2% of the total B cell population. The engineered B cells can be administered to a subject after it has been determined that a particular B cell clone in the final population does not constitute more than about 0.1% of the total B cell population, or more than about 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, or about 0.04% of the total B cell population. In certain embodiments, the engineered B cells (e.g., producing (follistatin)) are administered to a subject after it has been determined that a particular B cell clone in the final population does not constitute more than about 0.03% of the total B cell population.

[0182] In one embodiment, the cell composition is stored and / or shipped at 4°C. In another embodiment, the cell composition is frozen for storage and / or shipping. The cell composition can be frozen, for example, at -20°C or -80°C. In one embodiment, the step of freezing the cell composition comprises liquid nitrogen. In one embodiment, the cell composition is frozen using a controlled rate freezer. Thus, the methods described herein can further comprise a thawing step.

[0183] How to use One aspect of the present invention relates to the in vivo delivery of a therapeutic agent (e.g., follistatin) by the delivery of modified B cells engineered to express the therapeutic agent (e.g., follistatin). In certain embodiments, B cells modified to express follistatin are used in methods of treating and / or preventing chronic diseases and disorders and / or in methods of increasing muscle size or strength in a patient.

[0184] The modified B cells described herein can be administered in a manner appropriate to the disease or disorder to be treated or prevented.

[0185] The amount and frequency of administration will be determined by factors such as the condition of the patient and the type and severity of the patient's disease, but an appropriate dosage can be determined through clinical trials.

[0186] In one embodiment, a single dose of modified B cells is administered to the subject. In one embodiment, two or more doses of modified B cells are administered sequentially to the subject. In one embodiment, three doses of modified B cells are administered sequentially to the subject. In one embodiment, a dose of modified B cells is administered to the subject weekly, biweekly, monthly, bimonthly, quarterly, semi-annually, annually, or biennially. In one embodiment, when the amount of therapeutic agent produced by the modified B cells decreases, a second or subsequent dose of modified B cells is administered to the subject.

[0187] In one embodiment, a dose of modified B cells is administered to a subject at a certain frequency (e.g., weekly, biweekly, monthly, bimonthly, or quarterly) until a desired amount (e.g., an effective amount) of a therapeutic agent (e.g., follistatin) is detected in the subject. In one embodiment, the amount of the therapeutic agent (e.g., follistatin) is monitored in the subject. In one embodiment, if the amount of therapeutic agent produced by the modified B cells falls below the desired amount, a subsequent dose of modified B cells is administered to the subject. In one embodiment, the desired amount is in a range that produces a desired effect. For example, in a method for treating muscular dystrophy (e.g., Becker muscular dystrophy), the desired amount of follistatin can be the amount in the plasma of a subject receiving the modified B cells. In some embodiments, the desired amount can be the amount of follistatin that results in a certain level of weight gain by the subject. In some embodiments, the desired amount can be the amount of follistatin that results in a certain level of strength gain by the subject. In some embodiments, the desired amount can be the amount of follistatin that results in a certain level of weight gain in a subject.

[0188] When an "effective amount" or a "therapeutic amount" is indicated, the exact amount of the composition of the present disclosure to be administered can be determined by a physician, taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject). The B cell composition can also be administered multiple times at appropriate dose(s). The cells can be administered by using injection techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).

[0189] Optimal dosages and treatment regimens for a particular patient can be determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting treatment accordingly. Treatment can also be adjusted after measuring the level of a therapeutic agent (e.g., follistatin) in a biological sample (e.g., a body fluid such as plasma or a tissue sample) and used to assess treatment effectiveness, and treatment can be adjusted accordingly, either increased or decreased.

[0190] In some embodiments of the present disclosure, an optimal dose of engineered B cells for a multi-dose regimen can be determined by first determining an optimal single dose concentration of B cells for a subject, reducing the number of B cells present at the optimal single dose concentration to result in a suboptimal single dose concentration of engineered B cells, and administering two or more doses of the suboptimal single dose concentration of engineered B cells to the subject. In some embodiments, two, three, or more doses of the suboptimal single dose concentration of engineered B cells are administered to the subject. In some embodiments, administering two, three, or more doses of the suboptimal single dose concentration of engineered B cells to the subject results in a synergistic interaction of the therapeutic polypeptide that the engineered B cells are engineered to express. In some embodiments, the suboptimal single dose concentration comprises half, or 3, 4, 5, 6, 7, 8, 9, 10, or less than 1 / 10 of the optimal single dose concentration. In some embodiments, the therapeutic polypeptide is follistatin.

[0191] In some aspects of the present disclosure, smaller numbers of transfected B cells of the present disclosure can be administered, ranging from 10 cells per kilogram (10 to 10 cells per patient). In certain embodiments, B cells are administered to a subject at 1 x 10 cells, 5 x 10 cells, 1 x 10 cells, 5 x 10 cells, 1 x 10 cells, 5 x 10 cells, 1 x 10 cells, 5 x 10 cells, 1 x 10 cells, 5 x 10 cells, 1 x 10 cells, 5 x 10 cells, 1 x 10 cells, 5 x 10 cells, 1 x 10 cells, 5 x 10 cells, 1 x 10 cells, 5 x 10 cells, 1 x 10 cells, 5 x 10 cells, or 1 x 10 cells. B cell compositions can be administered multiple times at dosages within these ranges. Cells can be autologous or xenogeneic (e.g., allogeneic) to the patient receiving therapy. Optionally, treatment can also include administration of mitogens (e.g., PHA) or lymphokines, cytokines, and / or chemokines (e.g., GM-CSF, IL-4, IL-6, IL-13, IL-21, Flt3-L, RANTES, MIP1α, BAFF, etc.) described herein to enhance induction of an immune response and engraftment of the infused B cells.

[0192] Administration of the subject compositions can be carried out in any convenient manner, including by aerosol inhalation, injection, oral ingestion, infusion, implantation, or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intrathecally, intramuscularly, by intravenous (iv) injection, or intraperitoneally. The compositions described herein can be administered to a patient directly into the nervous system. In one embodiment, the B cell compositions of the present disclosure are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the B cell compositions described herein are preferably administered by iv injection. The B cell compositions can be injected directly into a tumor, lymph node, bone marrow, or site of infection.

[0193] In yet another embodiment, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, 1990, Science 249:1527-1533; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, a polymeric material can be used (Medical Applications of Controlled Release, 1974, Langer and Wise (eds.), CRC Press, Boca Raton, Fla.; Controlled Drug (See Bioavailability, Drug Product Design and Performance, 1984, Smolen and Ball (eds.), Wiley, New York; Ranger and Peppas, 1983; J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105.) In yet another embodiment, a controlled-release system can be placed in proximity to the therapeutic target, thereby requiring only a fraction of the systemic dose (see, e.g., Medical Applications of Controlled Release, 1984, Langer and Wise (eds.), CRC Press, Boca Raton, Fla., 2:115-138).

[0194] The B cell compositions of the present disclosure can also be administered using any number of matrices. Matrices have been utilized for many years within the context of tissue engineering (see, e.g., Principles of Tissue Engineering (Lanza, Langer, and Chick, eds.), 1997). The present disclosure utilizes such matrices within the novel context of acting as artificial lymphoid organs to support and maintain B cells. Thus, the present disclosure can utilize matrix compositions and formulations that have demonstrated utility in tissue engineering. Thus, the types of matrices that can be used in the compositions, devices, and methods of the present disclosure are virtually unlimited and can include both biological and synthetic matrices. In one particular example, compositions and devices described in U.S. Patent Nos. 5,980,889; 5,913,998; 5,902,745; 5,843,069; 5,787,900; or 5,626,561 are utilized. The matrix includes characteristics generally associated with being biocompatible when administered to a mammalian host. The matrix can be formed from natural and / or synthetic materials. The matrix can be non-biodegradable if it is desired to leave a permanent or removable structure within the animal's body, such as an implant; or it can be biodegradable. The matrix can take the form of a sponge, implant, tube, telfa pad, fiber, hollow fiber, lyophilized component, gel, powder, porous composition, or nanoparticle. Additionally, the matrix can be designed to allow for the sustained release of seeded cells or produced cytokines or other active agents. In certain embodiments, the matrix of the present disclosure can be described as a semi-solid scaffold that is flexible and elastic, and permeable to substances such as inorganic salts, aqueous fluids, and dissolved gaseous agents, including oxygen.

[0195] A matrix is ​​used herein as an example of a biocompatible material, however, the present disclosure is not limited to matrices, and thus, whenever the term matrix(ies) appears, such term should be construed to include devices and other materials that allow cell retention or traversal, are biocompatible, and can permit the traversal of macromolecules either directly through the material or used in conjunction with certain semipermeable materials such that the material itself is a semipermeable membrane.

[0196] In certain embodiments of the present disclosure, B cells transfected and activated using the methods described herein or other methods known in the art are administered to a patient in conjunction with (e.g., before, simultaneously with, or after) any number of relevant treatment modalities, including, but not limited to, treatment with drugs such as antivirals, chemotherapy, irradiation, immunosuppressants such as cyclosporine, bisulfin, bortezomib, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as campath, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. These drugs inhibit either the calcium-dependent phosphatase calcineurin (cyclosporine and FK506), the proteasome (bortezomib), or inhibit p70S6 kinase, which is important in growth factor-induced signal transduction (rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993; Isoniemi (see above). In further embodiments, the cell compositions of the present disclosure are administered to a patient in conjunction with (e.g., before, simultaneously with, or after) T cell depletion therapy using either bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or Campath. In one embodiment, the cell compositions of the present disclosure are administered after B cell depletion therapy using an agent that reacts with CD20, e.g., Rituxan®. In one embodiment, the cell compositions of the present disclosure are administered after B cell depletion therapy using an agent such as bortezomib. For example, in one embodiment, a subject can undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In certain embodiments, after transplantation, the subject receives an infusion of expanded immune cells of the present disclosure. In additional embodiments, the expanded cells are administered before or after surgery.

[0197] Dosages of the above-described treatments to be administered to patients will vary with the exact nature of the condition being treated and the recipient of the treatment. Scaling of dosages for human administration can be performed in accordance with art-accepted practice.

[0198] The engineered B cells can be used in the treatment or prevention of various diseases and disorders. In certain embodiments, B cells engineered to express follistatin are used in methods for increasing muscle size or strength in a patient. For example, the present disclosure provides a method for increasing muscle size or strength in a subject, comprising administering an effective amount of B cells expressing a follistatin polypeptide. The increase in muscle size or strength can occur generally throughout the subject or in targeted muscles. Muscular dystrophy (e.g., Duchenne muscular dystrophy, Becker muscular dystrophy, Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, stiff spine syndrome, Ullrich syndrome, Fukuyama muscular dystrophy, Walker-Warburg syndrome, muscle-eye-brain disease, facioscapulohumeral muscular dystrophy), congenital muscular dystrophy, myotonic dystrophy (Steiner's disease), non-dystrophic myotonia, periodic paralysis, and the like are examples of diseases that may be associated with the development of these conditions. Spinal muscular atrophy, familial amyotrophic lateral sclerosis, hereditary motor and sensory neuropathies, Charcot-Marie-Tooth disease, chronic inflammatory neuropathies, distal myopathies, myotubular / centronuclear myopathies, nemaline myopathies, minicore disease, centronuclear myopathies, desminopathies, inclusion body myositis, mitochondrial myopathies, congenital myasthenic syndromes, post-polio muscle dysfunction, and disorders described in Emery (2002) The Lancet, 359:687-695 and Khurana et al. (2003) Nat. Rev. Drug Disc., 2:379-386, inflammatory myopathies (e.g., inclusion body myositis, The targeted muscles may be damaged, weakened, or missing, as may be the case in various muscle disorders, including muscular disorders such as muscular atrophy (arthritis), muscle injury or trauma, muscle disuse (which may occur after prolonged bed rest or limb immobilization), and muscle atrophy or weakness as a result of aging, cancer, or various types of chronic diseases. For example, in some cases, muscles may be damaged, weakened, or missing due to sarcopenia. In some cases, muscles may be damaged, weakened, or missing due to spinal muscular atrophy (SMA). In some cases, muscles may be damaged, weakened, or missing due to amyotrophic lateral sclerosis (ALS). In some cases, muscles may be damaged, weakened, or missing due to Pompe disease. The method can also increase muscle size or strength in healthy muscles.

[0199] In some embodiments, the disclosure provides methods for treating a disease or disorder in an individual, the method comprising administering to a subject in need thereof B cells genetically modified to express follistatin (e.g., B cells expressing a follistatin+FST344 transcript variant).

[0200] In some embodiments, the disclosure provides methods for treating a myopathic disorder in an individual, the method comprising administering B cells genetically modified to express follistatin (e.g., B cells expressing a follistatin+FST344 transcript variant) to a subject having or suspected of having such myopathic disorder, wherein the myopathic disorder is muscular dystrophy. In some embodiments, the muscular dystrophies include Duchenne muscular dystrophy, Becker muscular dystrophy, Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, stiff spine syndrome, Ullrich syndrome, Fukuyama muscular dystrophy, Walker-Warburg syndrome, muscle-eye-brain disease, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, myotonic dystrophy (Steinert disease), non-dystrophic myotonia, periodic paralysis, spinal muscular atrophy, familial amyotrophic lateral sclerosis, hereditary motor and sensory neuropathies, Charcot-Marie-Tooth disease, chronic inflammatory neuropathies, distal myopathies, myotubular / centronuclear myopathy, nemaline myopathy, minicore disease, centronuclear disease, desminopathy, inclusion body myositis, mitochondrial myopathy, congenital myasthenic syndromes, post-polio muscle dysfunction, and Emery's syndrome. (2002) The Lancet, 359:687-695 and Khurana et al. (2003) Nat. Rev. Drug Disc., 2:379-386.

[0201] In some embodiments, the disclosure provides methods for treating a myopathic disorder in an individual, the method comprising administering B cells genetically modified to express follistatin (e.g., B cells expressing a follistatin+FST344 transcript variant) to a subject having or suspected of having such a myopathic disorder, wherein the myopathic disorder is an inflammatory myopathic disorder. In some embodiments, the inflammatory myopathic disorder is inclusion body myositis.

[0202] In some embodiments, the disclosure provides methods for treating a muscle disorder in an individual, the method comprising administering B cells genetically modified to express follistatin (e.g., B cells expressing a follistatin+FST344 transcript variant) to a subject having or suspected of having such a muscle disorder, wherein the muscle disorder is muscle injury or muscle trauma.

[0203] In some embodiments, the disclosure provides methods for treating a myopathic disorder in an individual, the method comprising administering B cells genetically modified to express follistatin (e.g., B cells expressing a follistatin+FST344 transcript variant) to a subject having or suspected of having such a myopathic disorder, wherein the myopathic disorder is muscle disuse (e.g., as may occur after prolonged bed rest or limb immobilization).

[0204] In some embodiments, the disclosure provides methods for treating a muscle disorder in an individual, the method comprising administering B cells genetically modified to express follistatin (e.g., B cells expressing a follistatin+FST344 transcript variant) to a subject having or suspected of having such a muscle disorder, wherein the muscle disorder is selected from muscle atrophy or weakness as a result of aging, cancer, or various types of chronic diseases.

[0205] In some embodiments, the disclosure provides methods for treating an individual who exhibits mild, moderate, or severe muscle weakness, muscle wasting, and / or effects on independent ambulation, comprising administering to the subject B cells genetically modified to express follistatin (e.g., B cells expressing a follistatin+FST344 transcript variant).

[0206] In some embodiments, the present disclosure provides methods for treating an individual exhibiting mild, moderate, or severe muscle weakness, muscle hypertrophy, muscle pseudohypertrophy, joint contractures, skeletal deformities, cardiomyopathy, dysphagia, bowel and bladder dysfunction, muscle ischemia, cognitive impairment, behavioral dysfunction, socialization dysfunction, scoliosis, and / or impaired respiratory function, the method comprising administering to the subject B cells genetically modified to express follistatin (e.g., B cells expressing a follistatin+FST344 transcript variant).

[0207] In certain embodiments, the present disclosure provides methods for treating muscular dystrophy in an individual, the method comprising administering B cells genetically modified to express follistatin (e.g., B cells expressing a follistatin+FST344 transcript variant) to a subject having or suspected of having Becker muscular dystrophy.

[0208] In some embodiments, a single maximally effective dose of follistatin+ B cells (e.g., follistatin+B cells expressing an FST344 transcript variant) is administered to a subject. In some embodiments, two or more doses of follistatin+B cells (e.g., follistatin+B cells expressing an FST344 transcript variant) are administered to a subject, thereby maximizing the amount of engrafted follistatin+B cells. In some embodiments, the two or more doses of follistatin+B cells (e.g., follistatin+B cells expressing an FST344 transcript variant) administered to a subject comprise fewer follistatin+B cells than a single maximally effective dose of follistatin+B cells. In some embodiments, when two or more doses of follistatin+ B cells (e.g., B cells expressing a follistatin+ FST344 transcript variant) are administered to a subject at a dosage of follistatin+ B cells that is less than the maximally effective single dose of follistatin+ B cells, a resulting synergistic increase in follistatin production occurs. In one embodiment, administering follistatin+ B cells to a subject results in normal levels of follistatin found in healthy control subjects. In one embodiment, administering follistatin+ B cells to a subject results in greater than normal levels of follistatin in the subject. In one embodiment, administering follistatin+ B cells (e.g., B cells expressing a follistatin+ FST344 transcript variant) to a subject increases the intensity of the subject. In one embodiment, administering follistatin+ B cells (e.g., B cells expressing a follistatin+ FST344 transcript variant) to a subject increases strength in the subject compared to normal levels. In one embodiment, administering follistatin+ B cells (e.g., B cells expressing a follistatin+ FST344 transcript variant) to a subject prevents loss of strength in the subject. [Example]

[0209] Example 1 Generation of follistatin-expressing B cells We generated Sleeping Beauty transposon and transposase constructs for transposition and expression of human follistatin (FST). The transposon was assembled to achieve FST gene integration and expression in B cells. We used the EEK promoter, which consists of promoter and enhancer elements from human immunoglobulin genes, as well as other previously described regulatory elements to achieve high-level expression in B cells. To test for FST transposition and expression, human B cells were isolated from two separate donors, expanded in culture in B cell culture medium, incubated at 37°C with 5% CO2, and electroporated with pKT2 / EEK-FST344 plus mRNA encoding the SB100x transposase on day 3. Cell lysates prepared 2, 5, 8, and 11 days after electroporation (i.e., on days 5, 7, 11, and 14 of culture) contained significantly increased FST compared with wild-type, untransfected cells, demonstrating the effectiveness of the SB transposon system for achieving high-level FST expression in expanded human B cells (Figure 5A). Notably, FST expression, after a slight decline from day 5 (likely due to early episomal FST expression), persisted from days 7 to 14, suggesting stable integration of the construct into B cells. Indeed, RT-PCR confirmed stable integration of the FST insert into the B cell genome (Figure 5B). Such FST-expressing B cells are referred to as FST+ B cells in the following examples.

[0210] Example 2 In vivo production of FST To determine whether B cells engineered according to the present disclosure can promote increased FST production in vivo, we injected the FST+ B cells generated in Example 1 into wild-type mice.

[0211] Specifically, four mice were treated on day 0 with either vehicle (500 μl phosphate-buffered saline (PBS)) or 2 × 10 IgG diluted in vehicle (PBS) to 500 μl. 6 3 × 10 human FST+ B cells were injected intravenously (tail vein) on day -7. Additionally, 3 × 10 human FST+ B cells were injected intravenously (tail vein) on day -7. 6 Primary autologous peripheral blood cells were injected intraperitoneally (ip) into mice to provide support for pKT2 / EEK-FST plus SB100x transposase-encoding mRNA-transposed B cells. We observed a two-fold increase in human FST in the plasma of mice treated with FST+ B cells, providing strong evidence of successful human B cell adoptive transfer (Figure 1). FST levels peaked approximately 28 days after injection and declined to near-normal levels by day 35. Although not performed in FST-deficient animals, the results of this experiment nevertheless provide an example of the levels of human FST that can be achieved after the introduction of a highly potent FST+ B cell population into wt mice. We found that plasma levels of FST correlated with plasma levels of human IgG, thus providing evidence of adoptive transfer and engraftment of FST+ B cells (Figures 2A-2D).

[0212] To determine whether FST+B cells had any effect on the treated mice, we monitored the body weight of control mice and mice treated with FST+B cells 35 days after injection. As shown in Figure 3, FST+B cells grew an average of 4.1% (0.9 grams) more than mice treated with vehicle control. Furthermore, weight gain corresponded to significant improvements in strength in the forelimb grip test (16% improvement in FST+B cell-treated mice compared to vehicle control) (Figure 4A); the limb grip test (23% improvement in FST+B cell-treated mice compared to vehicle control) (Figure 4B); and the hanging test (23% improvement in FST+B cell-treated mice compared to vehicle control) (Figure 4C).

[0213] Thus, such data demonstrate that B cells can be used in the methods disclosed herein to express and deliver FST to a subject to induce weight gain and improved strength.

[0214] The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referenced herein and / or listed in application data sheets are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified as necessary to employ concepts from the various patents, applications, and publications to provide further embodiments.

[0215] These and other changes can be made to the embodiments in light of the above detailed description. Generally, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments along with the full range of equivalents to which such claims are entitled. Accordingly, the claims are not limited by this disclosure.

Claims

1. A population of recombinant B cells having the following characteristics: i. at least 2 x 10 cells containing an exogenous follistatin gene operably linked to a promoter ６ at least 2 x 10 B cells, wherein the follistatin gene has been introduced into the B cells via transduction using a transposon system. ６ B cells; ii. when the population is administered to a subject, the population of B cells engraft in the subject and express the exogenous follistatin gene; and iii. Increase the follistatin protein level in the subject by at least 2-fold compared to the follistatin protein level in a subject not administered the population. A population of recombinant B cells comprising:

2. The population of recombinant B cells of claim 1 , wherein the promoter is an EEK promoter.

3. 2. The population of recombinant B cells of claim 1, wherein the transposon system is a Sleeping Beauty transposon system or a Piggybac transposon system.

4. 4. The population of recombinant B cells of claim 3, wherein the transposon system is the Sleeping Beauty transposon system.

5. The population of recombinant B cells of claim 1 , wherein the population exhibits a high degree of polyclonality.

6. 2. The population of recombinant B cells of claim 1, wherein any particular B cell clone in said population of recombinant B cells comprises less than 0.2% of said total B cell population.

7. 2. The population of recombinant B cells of claim 1, wherein any particular B cell clone in said population of recombinant B cells comprises less than 0.05% of said total B cell population.

8. 2. The population of recombinant B cells of claim 1, wherein the follistatin gene is a splice site variant of human follistatin FST-344.

9. 2. The population of recombinant B cells of claim 1, wherein the follistatin protein is secreted by the recombinant B cells.

10. 10. The population of recombinant B cells of claim 9, wherein the splice site variant of human follistatin FST-344 comprises SEQ ID NO: 3 or 4.

11. A composition comprising a population of recombinant B cells according to any one of claims 1 to 10 for use in a method of treating, preventing or alleviating a muscle disorder in a subject.

12. The composition of claim 11 , wherein the muscle disorder is a muscular dystrophy.

13. 13. The composition of claim 12, wherein the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, or facioscapulohumeral muscular dystrophy.

14. 12. The composition of claim 11, wherein the recombinant B cells are derived from B cells obtained from the subject, or the B cells are derived from cells obtained from the subject.

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