RU2832170C2 - B-cells, genetically modified for secretion of follistatin, and methods of using them for treating follistatin-related diseases, conditions, disorders and for increasing muscle growth and strength - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: invention relates to biotechnology and medicine, particularly to methods of introducing autologous and/or allogenic B-cells genetically modified for production of a drug such as follistatin.

EFFECT: cells and methods of the present invention can be used for long-term delivery of follistatin in vivo.

81 cl, 5 dwg, 2 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of 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, the entire contents of each of which are incorporated herein by reference.

POSITION REGARDING THE SEQUENCE LIST

[0002] The sequence listing associated with this application is provided in text format instead of a paper copy, and its contents are hereby incorporated by reference into this specification. The name of the text file containing the sequence listing is IMCO-008_01WO_ST25.txt. The text file is 12 KB, created on March 18, 2019, and received electronically via EFS-Web.

LEVEL OF TECHNOLOGY

Field of technology

[0003] The present invention relates to the use of B cells for long-term in vivo delivery of a drug such as follistatin and, in particular, to the administration of single and multiple doses of B cells to a subject (e.g., a human).

Description of the related area

[0004] Muscular dystrophies (MD) are progressive, inherited neuromuscular disorders characterized by muscle wasting and weakness (Emery (2002) The Lancet, 359:687-695). Many forms of muscular dystrophy are fatal and currently have no cure.

[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. Alteration or absence of this protein results in abnormal formation of gaps in the sarcolemmal membrane. Abnormal variability in muscle fiber diameter (atrophic and hypertrophic fibers) in proximal muscles and persistent muscle damage are characteristic features of the disease. Damaged muscle releases the intracellular enzyme creatine kinase (CK). As a result, serum CK levels in patients with DMD are characteristically high (up to 10 times higher than normal). The pathophysiological cascade combines tissue inflammation, muscle fiber necrosis, and muscle replacement with fibrofatty tissue.

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

[0007] Many pharmacological agents have been tried in MD, but none have been proven effective in stopping the progression of the disease. Current treatment is still in the realm of physical medicine and rehabilitation.

[0008] Several studies using corticosteroids (eg, prednisone and/or its derivatives) have shown 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, primarily affecting proximal muscles—the same muscles affected in DMD and BMD. Corticosteroid-induced muscle and other side effects may limit the long-term effectiveness of corticosteroid therapy.

[0009] The transforming growth factor-beta (TGF-beta) superfamily contains multiple growth factors that share common sequence elements and structural motifs. These proteins are known to exert biological effects on a wide variety of cell types in both vertebrates and invertebrates. Members of the superfamily perform important functions during embryonic development in pattern formation and tissue specification, and can influence a variety of differentiation processes including adipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis, and epithelial cell differentiation. The family is divided into two major clades, the BMP/GDF and the TGF-beta/activin/BMP10 clades, whose members exert diverse, often complementary, effects. By manipulating the activity of a TGF-beta family member, it is often possible to induce significant physiological changes in an organism. For example, Piedmontese and Belgian Blue cattle carry a loss-of-function mutation in the GDF8 gene (also called myostatin) that causes marked increases in muscle mass Grobet et al., Nat. Genet. 1997, 17(l):71-4. Furthermore, in humans, inactive alleles of GDF8 are associated with increased muscle mass and, reportedly, unusual strength. Schuelke et al., N Engl J Med 2004, 350:2682-8. Furthermore, mice genetically modified to express either the dominant negative activin IIB receptor (ActRIIB) or follistatin have remarkable muscle mass (Lee, SJ and McPherron, AC, Proc Natl Acad Sci USA. 2001 Jul 31;98(16):9306-11), and follistatin overexpression in nonhuman primates increases muscle growth and strength. 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 potent regulators of TGF-beta signaling.

[0011] Current treatments for chronic diseases and disorders include direct infusion of a drug (e.g., a therapeutic polypeptide), gene therapy via a viral vector, and adoptive transfer of stem cells (e.g., hematopoietic stem cell transfer). However, each of these methods has disadvantages. Injection of a recombinant therapeutic protein suffers from a finite protein half-life, and all three methods result in suboptimal tissue penetration of the drug. Alteration of endogenous tissues for drug production, such as through injection of recombinant adeno-associated virus (AAV) and lentiviral vectors, typically results in drug production from a centralized location. Drug production from a single location increases the chances of localized toxicity in the producing tissues. In addition, since recombinant viruses are considered foreign, it is unlikely that viral vectors can be administered multiple times without causing an adverse reaction, meaning that a single injection may be required to achieve the correct drug dose. Given the biological variability inherent in a method such as in vivo introduction of nucleic acids into cells using a virus, achieving the desired dose when limited to a single injection may be very variable.

[0012] Accordingly, there remains a need in the field for long-term treatment of many chronic diseases and disorders associated with TGF-beta signaling.

ESSENCE OF IMPLEMENTATION OPTIONS

[0013] The present invention relates generally to compositions and methods for administering and dosing genetically modified B cell compositions for the treatment of chronic diseases and disorders. In various embodiments, the present invention relates to compositions and methods for administering and dosing B cells genetically modified to express a polypeptide capable of modulating TGF-beta signaling (e.g., a follistatin polypeptide). In some specific embodiments, the present invention relates to compositions and methods for administering and dosing B cells genetically modified to express a follistatin polypeptide. In specific embodiments, the present invention 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 invention provides these and other advantages as described in the detailed description.

[0014] In some embodiments, the present invention provides a recombinant B cell 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 human follistatin splice site variant FST-344. In some embodiments, the B cell is a human B cell. In some embodiments, the B cell has been transduced with a follistatin gene. In some embodiments, the B cell comprises the follistatin gene because it has been transduced with the follistatin gene using the sleeping beauty transposon system. In some embodiments, the B cell expresses the follistatin gene due to transduction with a virus carrying the follistatin gene. In some embodiments, the B cell comprises a follistatin gene because it has been transduced with a retrovirus, lentivirus, adenovirus, or adeno-associated virus comprising a follistatin gene. In some embodiments, the B cell is modified to comprise a follistatin gene using a targeted integration method. In some embodiments, the targeted integration utilizes one or more zinc finger nucleases, transcription activator effector nuclease-like (TALEN) and/or CRISPR/Cas systems, including but not limited to CRISPR/Cas9 systems. In some embodiments, the B cell is modified to contain a follistatin gene by introducing a nucleic acid encoding follistatin using a method selected from the group consisting of introducing retroviral vectors, lentiviral vectors, adeno-associated viral vectors, adenoviral vectors, any other RNA or DNA viral vectors, non-viral DNA, and/or RNA encoding follistatin using chemical or physical methods such as lipofection, polycation complexation, electroporation, and the like. In some embodiments, the follistatin gene is secreted by a recombinant B cell. In some embodiments, the recombinant B cell is derived from a B cell derived from the subject or a B cell derived from a cell derived from the subject. In some embodiments, the recombinant B cell is derived from a B cell precursor derived from the subject. In some embodiments, the recombinant B cell is derived from a cell obtained from a subject that has differentiated into a B cell or a B cell precursor. In some embodiments, the recombinant B cell is modified by

(a) collecting and isolating immunocytes from the blood of a subject;

(b) transduction of cells with DNA encoding follistatin;

(c) ex vivo propagation of the selected cells; and

(d) differentiation of expanded cells ex vivo into plasma cells and/or plasmablasts.

[0015] In some embodiments, the isolated immune cells from step a are CD19 positive cells. In some embodiments, the transduction in step b is performed by electroporation. In some embodiments, the electroporation utilizes the sleeping beauty transposon system. In some embodiments, the differentiated cells are CD38(+) and CD20(-). In some embodiments, the present invention relates to a method comprising administering such a recombinant B cell to a subject.

[0016] In some embodiments, the present invention relates to a method of delivering follistatin to a subject in need thereof, comprising administering a recombinant B cell comprising a follistatin gene. In some embodiments, the present invention relates to a method of delivering follistatin to a subject in need thereof, comprising administering to the subject any of the recombinant B cells described herein expressing 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 muscle disorder. In some embodiments, the muscle disorder is muscular dystrophy. In some embodiments, the muscular dystrophy is selected from Duchenne muscular dystrophy, Becker muscular dystrophy, and facioscapulohumeral muscular dystrophy. In some embodiments, the muscle disorder is inflammatory muscle disorders. In some embodiments, the inflammatory muscle disorder is inclusion body myositis. In some embodiments, the muscle disorder is muscle injury or trauma. In some embodiments, the muscle disorder is muscle inactivity. In some embodiments, muscle inactivity occurs after prolonged bed rest or limb immobilization. In some embodiments, the muscle disorder is muscle atrophy or weakening. In some embodiments, the muscle atrophy or weakening is caused by aging, cancer, or chronic diseases. In some embodiments, the muscle atrophy or weakening is due to sarcopenia. In some embodiments, the muscle atrophy or weakening is due to spinal muscular atrophy (SMA). In some embodiments, the muscle atrophy or weakening is due to amyotrophic lateral sclerosis (ALS). In some embodiments, the muscle atrophy or weakening is due to Pompe disease. In some embodiments, the subject has healthy muscle. In some embodiments, administering a B cell expressing a follistatin polypeptide to a subject having healthy muscle increases the size or strength of the subject's muscles.

[0017] In some specific embodiments, the present invention provides a method of treating muscular dystrophy, comprising administering to a subject with muscular dystrophy a B cell expressing a follistatin polypeptide. In some embodiments, the subject has Becker muscular dystrophy. In some embodiments, administering a recombinant B cell to the subject provides a treatment for a disease, disorder, or condition of the subject. In some embodiments, administering a recombinant B cell to the subject provides a treatment for muscular dystrophy. In some embodiments, administering a recombinant B cell to the subject causes an increase in weight of the subject. In some embodiments, the subject's body weight increases by at least about 4%. In some embodiments, a significant increase in body weight occurs within 30 days. In some embodiments, a significant increase in body weight occurs within about 30 days. In some embodiments, administering a recombinant B cell to the subject causes an increase in muscle mass in the subject. In some embodiments, administering a recombinant B cell to a subject causes an increase in the subject's strength. In some embodiments, administering a recombinant B cell causes an increase in follistatin levels in the subject's plasma.

[0018] In some embodiments, the present invention provides a method of treating, preventing, or ameliorating muscular dystrophy by administering a recombinant B cell comprising a follistatin gene. In some embodiments, the method of treating, preventing, or ameliorating muscular dystrophy comprises administering any of the recombinant B cells described 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 administration comprises two or more doses of genetically modified B cells at suboptimal single dose concentrations. In some embodiments, the administration 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 a 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 administration comprises intravenous, intraperitoneal, subcutaneous, or intramuscular injection. In some embodiments, the administration comprises intravenous injection. In some embodiments, the genetically modified B cells are modified on day 2 or day 3 after culture. In some embodiments, the genetically modified B cells are modified using a method comprising electroporation. In some embodiments, the genetically modified B cells are harvested for administration to the subject on day 4, day 5, day 6, or day 7 in culture after modification. In some embodiments, the genetically modified B cells are harvested for administration to the subject on or after day 8 in culture after modification. In some embodiments, the genetically modified B cells are harvested for administration to the subject on or before day 10 in culture after modification. In some embodiments, the harvested genetically modified B cells do not produce significant levels of proinflammatory cytokines. In some embodiments, the genetically modified B cells are harvested at a time point in culture at which they are determined not to produce significant levels of proinflammatory cytokines. In some embodiments, 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 culture period, before and after modification. In some embodiments, the multimerized CD40 ligand is a HIS-tagged CD40 ligand multimerized using an anti-his antibody. In some embodiments, the method further comprises expanding the genetically modified B cells prior to administration 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 clone of B cells in the final population of expanded genetically modified B cells is less than 0.2% of the total B cell population. In some embodiments, any particular clone of B cells in the final population of expanded genetically modified B cells is 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 with enhanced methotrexate resistance. In some embodiments, the human DHFR gene with increased methotrexate resistance comprises leucine to tyrosine mutations at amino acid position 22 and phenylalanine to serine mutations at amino acid position 31. In some embodiments, the method comprises treating the genetically modified B cells with methotrexate prior to harvesting for administration. In some embodiments, the methotrexate treatment is between 100 nM and 300 nM. In some embodiments, the methotrexate treatment is at 200 nM. In some embodiments, the genetically modified B cells migrate to various tissues when administered to the subject. In some embodiments of the method, at least one genetically modified B cell from the population of genetically modified B cells that are 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 from a population of genetically modified B cells that are 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 relates to a modified B cell transduced to express both a follistatin gene and a dihydrofolate reductase (DHFR) gene.

BRIEF DESCRIPTION OF DRAWINGS

[0020] 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 treated groups: bars correspond to days 21, 28, and 35, respectively.

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

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

[0023] Figure 4 shows strength assessments in mice treated or not treated with follistatin-expressing B cells. FIG. 4A shows strength as assessed by the forepaw elevated bar walk test. FIG. 4B shows strength as assessed by the quadruped elevated bar walk test. FIG. 4C shows strength as assessed by the suspension test. The percentage improvements shown below the graphs indicate the average percentage improvement in the treated group compared to the untreated group.

[0024] Figure 5 shows in vitro expression of follistatin in follistatin-expressing B cells. FIG. 5A shows follistatin protein expression as determined by ELISA. FIG. 5B shows follistatin mRNA expression as determined by RT-PCR.

DETAILED DESCRIPTION

[0025] The practice of the present invention will employ, unless otherwise specifically indicated, conventional techniques of molecular biology, recombinant DNA, protein expression, and protein/peptide/carbohydrate chemistry, within the skill of the art, many of which are described below for illustrative purposes. Such techniques are fully explained in the literature. See, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2000); DNA Cloning: A Practical Approach, vol. 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 & S. Higgins, eds., 1985); Nucleic Acid Hybridization: Modern Applications (Buzdin and Lukyanov, eds., 2009); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Freshney, RI (2005) Culture of Animal Cells, a Manual of Basic Technique, 5th Ed. Hoboken NJ, John Wiley &Sons; B. Perbal, A Practical Guide to Molecular Cloning (3rd Edition 2010); Farrell, R., RNA Methodologies: A Laboratory Guide for Isolation and Characterization (3rd Edition 2005). The publications discussed above are provided only for their disclosure prior to the filing date of the present application. Nothing in this specification should be construed as an admission that the inventors are not entitled to antedate such specification because of prior invention.

DEFINITIONS AND ABBREVIATIONS

[0026] Unless defined otherwise, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the specification and the appended claims, unless otherwise defined, the following terms have the specified meaning. For the purposes of the specification, whenever the definition of a term as defined in this specification differs from the definition given for the same term in an incorporated reference, the definition expressly defined in this specification is the correct definition of that term.

[0027] The singular forms denote one or more unless otherwise specifically stated.

[0028] By "about" is meant a quantity, level, magnitude, number, frequency, percentage, measurement, size, value, mass, or length that differs by up to 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% from a reference quantity, level, magnitude, number, frequency, percentage, measurement, size, value, mass, or length. In any embodiment discussed 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" may comprise an active agent and a carrier, inert or active, such as a pharmaceutically acceptable carrier, diluent, or excipient. In particular embodiments, the compositions are sterile, substantially endotoxin-free, or nontoxic to recipients at the dose or concentration employed.

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

[0031] By "consisting of" is meant including and limited to whatever follows the expression "consisting of." Thus, the expression "consisting of" indicates that the listed elements are necessary or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the expression, and limited to other elements that do not interfere with or contribute to the activity or action specified in the description for the listed elements. Thus, the expression "consisting essentially of" indicates that the listed elements are necessary or mandatory, but that other elements are optional and may or may not be present, depending on whether they affect the activity or action of the listed elements.

[0032] Reference throughout the present specification to "biological activity" or "bioactivity" refers to any response induced in an in vitro assay or in a cell, tissue, organ, or organism (e.g., an animal or mammal, or a human) by administration of any compound, agent, polypeptide, conjugate, pharmaceutical composition provided herein. Biological activity may refer to agonistic actions or antagonistic actions. Biological activity may be a beneficial effect; or biological activity may not be beneficial, i.e., toxicity. In some embodiments, biological activity may refer to the positive or negative effects that a drug or pharmaceutical composition has on a living subject, e.g., a mammal, such as a human. Accordingly, the term "biologically active" is intended to describe any compound having biological activity as described herein. Biological activity can be assessed by any suitable methods currently known to one of skill in the art. Such assays may be qualitative or quantitative. The need to use different assays to assess the activity of different polypeptides is well understood by those skilled in the art; this is a routine task for the average researcher. Such assays are often easily performed in the laboratory with little need for optimization, and in most cases, commercial kits are available that provide simple, reliable, and reproducible readings of biological activity for a wide range of polypeptides using a variety of technologies commonly found in laboratories. When such kits are not available, average researchers can easily develop and optimize in-house bioactivity assays for target polypeptides without undue experimentation; this is a routine aspect of the scientific process.

[0033] Reference to the term "for example" is intended to mean "for example, but without limitation," and thus it should be understood that what follows is merely an example of a particular embodiment, but should in no way be considered a limiting example. Unless otherwise indicated, the use of "for example" is intended to clearly indicate that other embodiments are contemplated by and included in the present invention.

[0034] Reference throughout this specification to "an embodiment" or "one embodiment" or "an embodiment" or "some embodiments" or "particular embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearance of the phrases "in one embodiment" or "in an embodiment" or "in particular embodiments" in various places throughout this specification does 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 may include an increase that is 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 times (e.g., 100, 500, 1000 times) (including all integers and decimal fractions therebetween and above 1, e.g., 2.1, 2.2, 2.3, 2.4, etc.) of the amount or level described herein. Similarly, a "reduced" or "lowered" or "lesser" amount typically represents a "statistically significant" amount, and may include a decrease that is 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 times (e.g., 100, 500, 1000 times) (including all integers and decimal fractions therebetween and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the amount or level described herein.

[0036] The terms " in vitro ", " ex vivo " and " in vivo " are intended herein to have their normal scientific meanings. Accordingly, for example, " in vitro " is intended to refer to experiments or reactions occurring with isolated cellular components, such as, for example, an enzymatic reaction carried out in a test tube using an appropriate substrate, enzyme, donor and optionally buffers/cofactors. " Ex vivo " is intended to refer to experiments or reactions carried out using functional organs or cells removed from the organism or propagated independently of the organism. " In vivo " is intended to refer to experiments or reactions carried out within a living organism in its normal intact state.

[0037] "Mammal" includes humans and both domestic animals, such as laboratory animals and pets (e.g., cats, dogs, pigs, cattle, sheep, goats, horses, and rabbits), and non-domestic animals, such as wild animals, etc.

[0038] “Optional” or “optional” means that the event or condition then described may or may not occur, and that the description includes instances in which the stated event or condition occurs and instances in which it does not occur.

[0039] "Pharmaceutical composition" refers to a composition of a compound (e.g., a therapeutically useful polypeptide) and a vehicle conventional in the art for delivering the compound to an animal, such as a human. Such a vehicle may thus include any pharmaceutically acceptable carriers, diluents, or excipients.

[0040] "Pharmaceutically effective excipients" and "pharmaceutically effective carriers" are well known to those skilled in the art, and methods for their preparation are also 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, the contents of which are incorporated herein by reference).

[0041] The terms "polynucleotide", "nucleotide", "nucleotide sequence" and "nucleic acid" are used interchangeably. They refer to the polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, locus(es) determined 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. The polynucleotide may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be introduced before or after polymer assembly. The nucleotide sequence may include non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0042] "Subject" within the scope of the invention includes any animal that exhibits a disease or symptom, or that is at risk of exhibiting a disease or symptom that can be treated using the agent of the invention. Suitable subjects include laboratory animals (such as a mouse, rat, rabbit or guinea pig), farm animals and domestic animals or pets (such as a cat or dog). Also included are non-human primates and, preferably, human patients.

[0043] "Substantially" or "substantially" means more than a sufficient or substantial number, quantity, amount; nearly total or complete; for example, 95% or more of some given quantity.

[0044] “Medicine” refers to any compound that, when administered to a subject (e.g., preferably a mammal, more preferably a human) in a therapeutically effective amount, is capable of effectively treating a disease or condition as defined below.

[0045] "A therapeutically effective amount" or "a therapeutically effective dose" refers to an amount of a compound of the invention that, when administered to a subject (e.g., preferably a mammal, more preferably a human), is sufficient to provide treatment, as defined below, for a disease or condition in an animal. The amount of a compound of the invention constituting a "therapeutically effective amount" may vary depending on the compound, the condition and its severity, the route of administration, and the age of the animal being treated, but can be determined in a manner routinely by a person skilled in the art, based on his or her own knowledge and this disclosure.

[0046] "Treatment" or "treatment," as used herein, includes treating a disease or condition of interest in a subject, preferably a human, having the disease or condition of interest, and includes: (i) preventing or inhibiting the onset of the disease or condition in the subject, particularly where such subject is predisposed to the condition but has not yet been diagnosed as having it; (ii) inhibiting the disease or condition, i.e., stopping its progression; (iii) alleviating the disease or condition, i.e., causing regression of the disease or condition; or (iv) alleviating symptoms resulting from the disease or condition. Within the scope of the invention, the terms "disease", "disorder" and "condition" may be used interchangeably, or they may differ in that a particular disease, injury or condition may have no known etiologic agent (so that the etiology has not yet been developed), and they are thus not yet recognized as an injury or disease, but only as an undesirable condition or syndrome, where a more or less specific set of symptoms has been identified by clinicians.

REVIEW

[0047] The present invention relates, among other things, to autologous and/or allogeneic B cells altered by the administration of nucleic acids to produce follistatin, and also relates to methods of administering the modified B cells (e.g., to treat a disease, disorder, or condition, such as a muscle disorder such as muscular dystrophy). In some embodiments, the terms "engineered B cell,""genetically engineered B cell,""modified B cell," and "genetically modified B cell" are used interchangeably herein to refer to such altered B cells comprising one or more nucleic acids (e.g., a transgene) to produce follistatin (e.g., a transgene that provides for the expression of a follistatin polypeptide, such as a therapeutic follistatin polypeptide). Specifically, the modified B cells can be administered in the form of a single dose or multiple doses.

[0048] Accordingly, the methods of administering the modified B cell compositions described herein can be used for long-term in vivo delivery and expression of follistatin. The present invention relates generally to methods for achieving sufficient enrichment and quantity of follistatin-producing cells and sufficient levels of follistatin in vivo while ensuring product safety.

[0049] As used herein, the terms "long-term in vivo survival" and "long-term survival" refer to the survival of the modified B cells described herein for 10 or more days after administration to a subject. Long-term survival may be measured in days, weeks, or even years. In one embodiment, a majority of the 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 or more days after administration. In one embodiment, a 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 or more weeks 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 or more years. Furthermore, while the modified B cells described herein can survive in vivo for 10 days or more, it is understood that most modified B cells survive in vivo for 1, 2, 3, 4, 5, 6, 7, 8, 9 or more days after administration. Accordingly, it is envisaged that the modified B cells described herein may be used in short-term treatment (e.g., 4 days) and long-term treatment (e.g., 30 days or more) methods.

B cells

[0050]After leaving the bone marrow, the B cell acts as an antigen-presenting cell (APC) and internalizes antigens. The antigen is taken up by the B cell via receptor-mediated endocytosis and processed. The antigen is processed to antigenic peptides, loaded onto MHC II molecules, and presented on the extracellular surface of the B cell to CD4+ helper T cells. These T cells bind to the MHC II molecule/antigen and induce B cell activation. Following stimulation by the T cell, the activated B cell begins to differentiate into more specialized cells. Germinal center B cells can differentiate into long-lived memory B cells or plasma cells. In addition, secondary immune stimulation can cause memory B cells to give rise to additional plasma cells. The formation of plasma cells from either memory B cells or non-memory B cells is preceded by the formation of plasmablast precursors, which ultimately differentiate into plasma cells that produce large amounts of antibody (see, e.g., Trends Immunol. 2009 June; 30(6): 277–285; Nature Reviews, 2005, 5:231–242). Plasmablasts secrete more antibody than B cells but less than plasma cells. They divide rapidly, and they continue to internalize antigens and present antigens to T cells. Plasmablasts have the ability to migrate to sites of chemokine production (e.g., in the bone marrow), whereby they can undergo differentiation into long-lived plasma cells. Ultimately, the plasmablast can either remain in the plasmablast form for several days and then die, or undergo irreversible differentiation into a mature, fully differentiated plasma cell. Specifically, plasmablasts capable of homing to tissues containing niches for plasma cell survival (e.g., bone marrow) are able to replace their inhabiting plasma cells to become long-lived plasma cells that can continue to secrete high levels of proteins for several years.

[0051] B cells used in the methods described herein (e.g., to express follistatin) 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 conventional pan-B cell markers such as CD19 and CD20 and express relatively few surface antigens. Plasma cells express CD38, CD78, CD138, and the interleukin-6 receptor (IL-6R), and lack expression of CD45, and these markers can be used, such as by flow cytometry, to identify plasma cells. CD27 is also a good marker for plasma cells, since naive B cells are CD27-, memory B cells are CD27+, and plasma cells are CD27++. Subsets of memory B cells can also express surface IgG, IgM, and IgD, while plasma cells do not express these markers on the cell surface. CD38 and CD138 are expressed at high levels on plasma cells (see Wikipedia, The Free Encyclopedia., “Plasma cell” Page Version ID: 404969441; Date of last revision: 30 December 2010 09:54 UTC, retrieved January 4, 2011; see also: Jourdan et al. Blood. 2009 Dec 10;114(25):5173–81; Trends Immunol. 2009 June; 30(6): 277–285; Nature Reviews, 2005, 5:231–242; Nature Med. 2010, 16:123–129; Neuberger, M. S.; 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, 2-Vol. Set. Hagerstwon, MD: Lippincott Williams & Wilkins. pp. 347; Walport, Mark; Murphy, Kenneth; Janeway, Charles; Travers, Paul J. (2008). Janeway's immunobiology. New York: Garland Science, pp. 387-388; Rawstron AC (May 2006). "Immunophenotyping of plasma cells". Curr Protoc Cytom).

[0053] “Dormant,” as used herein, refers to a state of cells where the cell is not actively proliferating.

[0054] “Activated,” as used herein, refers to a state of a cell wherein the cell is actively proliferating and/or producing cytokines in response to a stimulus.

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

[0056] The term "subject" is intended to include living organisms in which an adaptive immune response can be elicited (e.g., mammals). 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 variety of sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, cord blood, tissue from an infection site, spleen tissue, and tumors. In a preferred embodiment, the source of B cells is PBMC. In particular embodiments of the present invention, any number of B cell lines available in the art can be used.

[0057] In particular embodiments of the methods described herein, B cells can be obtained from a unit of blood collected from a subject using any number of methods known to one of skill in the art, such as separation on Ficoll ^™ (sucrose-epichlorohydrin copolymers that can be used to produce high-density solutions). In one preferred embodiment, cells are obtained from the circulating blood of an individual by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. In one embodiment, the cells collected by apheresis can be washed to remove the plasma fraction and to place the cells in a suitable 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 an alternative embodiment, the wash solution lacks calcium and may lack magnesium, or may lack many, if not all, divalent cations. As is well known to one of skill in the art, the washing step can be performed by methods known in the art, such as 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, for example, PBS. Alternatively, unwanted components can be removed from the apheresis sample and the cells resuspended directly in the culture medium.

[0058] B cells can be isolated from peripheral blood or leukapheresis product using methods known in the art. For example, PBMC can be isolated using Ficoll ^™ (Sigma-Aldrich, St Louis, MO) and CD19+ B cells 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 particular embodiments, memory B cells are isolated as described in Jourdan et al., (Blood. 2009 Dec 10; 114(25):5173-81). For example, after removing CD2+ cells using anti-CD2 magnetic beads, CD19+ CD27+ memory B cells can be sorted by FACS. Bone marrow plasma cells (BMPCs) can be purified using anti-CD138 magnetic bead sorting or other similar methods and reagents. Human B cells can be isolated, for example, using anti-human CD19 microbeads (Miltenyi Biotec, San Diego, CA). Human memory B cells can be isolated, for example, using a human memory B cell isolation kit (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 particular embodiments, resting B cells can be obtained by sedimentation in continuous Percoll gradients as described in (Defranco et al., (1982) J. Exp. Med. 155:1523).

[0060] In one embodiment, PBMCs are obtained from a blood sample using a gradient-based purification (e.g., Ficoll™). In another embodiment, PBMCs are obtained by 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 includes depletion of T cells using anti-CD3 antibody-conjugated microbeads, thereby obtaining a T cell-depleted fraction. In a further embodiment, memory B cells are isolated from the pan-B cells or T cell-depleted fraction by positive selection for CD27.

[0061] In one specific embodiment, memory B cells are isolated by depletion of unwanted cells and subsequent positive selection using anti-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. A "switched memory B cell" or "switched B cell," as used herein, refers to a B cell that has undergone isotype class switching. In one embodiment, the switched memory B cells are positively selected for IgG. In another embodiment, the switched memory B cells are obtained by depletion of IgD and IgM expressing cells. The switched memory B cells can be isolated, for example, using a human switched memory B cell isolation kit (Miltenyi Biotec, San Diego, CA).

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

[0064] In a further embodiment, a promoter sequence from a gene unique to memory B cells, such as, for example, the CD27 gene (or another gene specific to memory B cells and not expressed in naive B cells), is used to control the expression of a selectable marker, such as, for example, a mutant dihydrofolate reductase that allows 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, for example, the CD19 gene, is used to control the expression of a selectable marker, such as, for example, a mutant dihydrofolate reductase that allows positive selection of memory B cells in the presence of methotrexate. In another embodiment, the 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 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 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.

B cell culture in vitro

[0065] B cells, such as memory B cells, can be cultured using in vitro methods to activate and differentiate B cells into plasma cells or plasmablasts, or both. As known to one of skill in the art, plasma cells can be identified by patterns of cell surface protein expression using standard flow cytometric 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 absence of CD45. CD27 can also be used to identify plasma cells, since 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, the B cells are contacted with one or more B cell activating factors, such as 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 1 13:1422-1431). Such factors may be selected from the group consisting of, but not limited to, 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, the C-type chemokines XCL1 and XCL2, the CC-type chemokines (currently including CCL1-CCL28) and the CXC-type chemokines (currently including CXCL1-CXCL17), and members of the TNF superfamily (e.g., TNF-α, BB ligand 4-1, B cell activating factor (BLyS), FAS ligand, sCD40L (including multimeric variants of sCD40L; e.g., histidine-tagged soluble recombinant CD40L in combination with anti-polyhistidine mAb to co-assemble multiple sCD40L molecules), lymphotoxin, OX40L, RANKL, TRAIL), CpG and other toll-like receptor agonists (e.g., CpG).

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

[0071] For example, in some embodiments, B cells are cultured using a B cell culture medium comprising one or more B cell activating factors selected from CD40L, IL-2, IL-4, and IL-10 to expand the B cells. In one embodiment, the B cells are cultured with 0.25-5.0 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, the CD40L molecules are covalently cross-linked or held together using protein multimerization domains (e.g., an IgG Fc region 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, the B cells are cultured with 1-10 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 1-10 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 10-100 ng/ml IL-10. In one embodiment, the concentration of IL-10 is 40 ng/ml.

[0072] In one embodiment, B cells are cultured using a B cell culture medium comprising one or more B cell activating factors selected from CD40L, IL-2, IL-4, IL-10, IL-15, and IL-21 to expand the B cells. In one embodiment, the B cells are cultured with 0.25-5.0 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, the CD40L molecules are covalently cross-linked or held together using protein multimerization domains (e.g., an IgG Fc region 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 1-10 ng/ml IL-2. In one embodiment, the concentration of IL-2 is 5 ng/ml. In one embodiment, B cells are cultured with 1-10 ng/ml IL-4. In one embodiment, the concentration of IL-4 is 2 ng/ml. In one embodiment, B cells are cultured with 10-100 ng/ml IL-10. In one embodiment, the concentration of IL-10 is 40 ng/ml. In one embodiment, B cells are cultured with 50-150 ng/ml IL-15. In one embodiment, the concentration of IL-15 is 100 ng/ml. In one embodiment, the B cells are cultured with 50-150 ng/ml IL-21. In one embodiment, the IL-21 concentration is 100 ng/ml. In a specific embodiment, the B cells are cultured using a B cell culture medium comprising CD40L, IL-2, IL-4, IL-10, IL-15, and IL-21 to expand the B cells.

[0073] For example, in one embodiment, B cells are cultured using a B cell culture medium comprising the B cell activating factors CD40L, IL-2, IL-4, IL-10, IL-15, and IL-21 to expand the B cells, wherein CD40L is cross-linked using a cross-linking agent to produce CD40L multimers. Such a culture system can be maintained throughout a culture period (e.g., a 7-day culture period) in which the B cells are transfected or otherwise modified to express a transgene of interest (e.g., an exogenous polypeptide, such as, for example, FST). The transgene can be integrated into the B cell (e.g., via a viral or non-viral vector). The transgene can be expressed in the B cell using a transposon. The transgene can be expressed in a B cell by targeted integration of the transgene into the B cell genome. Targeted integration can be achieved by homologous recombination. Homologous recombination can occur at a double-strand break induced by a nuclease. The nuclease can be, for example, a zinc finger nuclease, a TALE nuclease (TALEN), a meganuclease (e.g., a homing endonuclease), or the CRISPR/CAS9 nuclease system.

[0074] In another example, in one embodiment, the B cells are cultured using a B cell culture medium comprising 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) to differentiate the B cells. In one embodiment, the B cells are cultured with 25-75 ng/mL CD40L. In one embodiment, the concentration of CD40L is 50 ng/mL. In one embodiment, the B cells are cultured with 250-750 U/mL IFN-α. In one embodiment, the concentration of IFN-α is 500 U/mL. In one embodiment, the B cells are cultured with 5-50 U/mL IL-2. In one embodiment, the concentration of IL-2 is 20 U/mL. In one embodiment, the B cells are cultured with 25-75 ng/mL IL-6. In one embodiment, the concentration of IL-6 is 50 ng/mL. In one embodiment, the B cells are cultured with 10-100 ng/mL IL-10. In one embodiment, the concentration of IL-10 is 50 ng/mL. In one embodiment, the B cells are cultured with 1-20 ng/mL IL-15. In one embodiment, the concentration of IL-15 is 10 ng/mL. In one embodiment, the B cells are cultured with 10-100 ng/mL IL-21. In one embodiment, the concentration of IL-21 is 50 ng/mL. In one embodiment, the B cells are cultured with 1-50 μg/mL p-ODN. In one embodiment, the concentration of p-ODN is 10 μg/mL.

[0075] In one embodiment, the B cells are contacted with or cultured on feeder cells. In one embodiment, the feeder cells are a stromal cell line, such as a murine stromal cell line S17 or MS5. In another embodiment, the isolated CD19+ cells are cultured with one or more B cell activating factor 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 culture plate or a bead. In another embodiment, purified B cells are cultured, in the presence or absence of feeder cells, 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.

[0076] In another embodiment, B cell activation factors are provided by transfection of a B cell or other feeder cell. In this context, one or more factors that stimulate differentiation of a B cell into an antibody-secreting cell and/or one or more factors that promote the survival of an antibody-producing cell can be used. Such factors include, for example, Blimp-1, TRF4, anti-apoptotic factors like Bcl-xl or Bcl5, or constitutively active mutants of the CD40 receptor. In addition, factors that stimulate expression of downstream signaling molecules, such as TNF receptor-associated factors (TRAFs), can also be used in B cell activation/differentiation. In this regard, cell activation, cell survival, and antiapoptotic functions of the TNF receptor superfamily are largely mediated by TRAF1-6 (see, e.g., R. H. Arch, et al., Genes Dev. 12 (1998), pp. 2821-2830). Downstream effectors of TRAF signaling include the NF-κB and AP-1 family of transcription factors, which can mediate the switching on of genes involved in various aspects of cellular and immune function. Furthermore, activation of NF-κB and AP-1 has been shown to protect cells from apoptosis through the transcription of antiapoptotic genes.

[0077] In another embodiment, Epstein-Barr virus (EBV)-derived proteins are used to activate and/or differentiate B cells, or to promote the survival of an antibody-producing cell. EBV-derived proteins include, but are 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 particular embodiments, contacting B cells with B cell activating factors using the methods described herein results in, among other things, proliferation (i.e., expansion) of the cells, modulation of the IgM+ cell surface phenotype to a phenotype consistent with an activated mature B cell, 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 particular embodiments, CD40L fibroblasts are irradiated prior to use in the methods described herein. In one embodiment, the 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 particular embodiments, the methods comprise culturing the B cells in the presence of one or more of the aforementioned factors in combination with transformed stromal cells (e.g., MS5) that provide low levels of anchored CD40L and/or plate- or bead-bound CD40L.

[0079] As discussed above, B cell activating factors induce expansion, proliferation, or differentiation of B cells. Accordingly, B cells are contacted with one or more of the B cell activating factors listed above to produce an expanded cell population. The cell population can be expanded prior to transfection. Alternatively or additionally, the cell population can be expanded after transfection. In one embodiment, expanding 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, expanding the B cell population comprises culturing the cells with IL-2, IL-10, CpG, and CD40L. In one embodiment, expanding the B cell population comprises culturing the cells with IL-2, IL-4, IL-10, IL-15, IL-21, and CD40L. In one embodiment, expanding 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 the B cell population is induced and/or enhanced by a transgene introduced into the B cells. For example, a B cell comprising a recombinant receptor or an engineered receptor that induces a cellular signaling pathway (e.g., signaling downstream of CD40) upon binding its ligand (e.g., a soluble ligand or a cell surface expressed ligand). In one embodiment, the B cell overexpresses CD40 due to expression of a CD40 transgene. In another embodiment, the B cell expresses 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 an intracellular signaling portion of a B cell receptor (e.g., CD40).

[0081] In one embodiment, expansion of a B cell population is induced and/or enhanced by a small molecule compound added to the cell culture. For example, a compound that binds and dimerizes CD40 can be used to trigger the CD40 signaling pathway.

[0082]Any of a variety of culture media can be used in the present methods, as known to one of skill in the art (see, e.g., Current Protocols in Cell Culture, 2000-2009 by 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 (in the presence or absence of fetal bovine or other appropriate serum). Exemplary media also include, but are not limited to, IMDM, RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20. In further embodiments, the medium can comprise a surfactant, an antibody, a plasmanate, or a reducing agent (e.g., N-acetylcysteine, 2-mercaptoethanol), one or more antibiotics, and/or additives such as insulin, transferrin, sodium selenite, and cyclosporine. In some embodiments, IL-6, soluble CD40L, and a cross-linking enhancer may also be used.

[0083] The B cells are cultured under conditions and for sufficient periods of time to achieve the desired differentiation and/or activation. In particular embodiments, the B cells are cultured under conditions and for sufficient periods 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, the B cells are activated and differentiated to a mixed population of plasmablasts and plasma cells. As will be appreciated by one of skill in the art, plasmablasts and plasma cells can be identified by cell surface protein expression patterns using standard flow cytometric methods as described elsewhere herein, such as expression of one or more of CD38, CD78, IL-6R, CD27 ^high , and CD138, and/or absence or decreased expression of one or more of CD19, CD20, and CD45. As will be appreciated by one of skill in the art, memory B cells are typically 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, the cells can be cultured under suitable 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 or more days. The cells are reseeded, and media and additives can be added or changed as needed, using methods known in the art.

[0084] In particular embodiments, the B cells are cultured under conditions and for sufficient periods 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 for Ig production and/or transgene expression.

[0085] Induction of B cell activation can be measured by methods such as ³ H-uridine incorporation into RNA (as B cells differentiate, RNA synthesis increases) or by ³ H-thymidine incorporation, which measures DNA synthesis associated with cell proliferation. In one embodiment, interleukin-4 (IL-4) can be added to the culture medium at an appropriate concentration (e.g., about 10 ng/mL) to enhance B cell proliferation.

[0086] Alternatively, B cell activation is measured as a function of immunoglobulin secretion. For example, CD40L is added to resting B cells along with IL-4 (e.g., 10 ng/ml) and IL-5 (e.g., 5 ng/ml) or other B cell activating cytokines. 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; 5:1 -16; Loken, MR, et al, Flow Cytometry Characterization of Erythroid, Lymphoid and Monomyeloid Lineages in Normal Human Bone Marrow, in Flow Cytometry in Hematology, Laerum OD, Bjerksnes R. eds., Academic Press, New York 1992; pp. 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, or more days, typically about 3 days, additional volume of culture medium can be added. Supernatant from individual cultures can be collected at various times during the culture and quantified for IgM and IgG1 as described in Noelle et al., (1991) J. Immunol. 146:1118-1124. In one embodiment, the culture is collected and measured for expression of the transgene of interest using flow cytometry, enzyme-linked immunosorbent assay (ELISA), ELISPOT, or other assay known in the art.

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

[0089] In particular embodiments, the B cells are cultured under conditions and for sufficient periods 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 times or more greater than the number of B cells at the start of the culture. In one embodiment, the number of cells is 10-1000 times greater, including successive integers therein, than the number of B cells at the start of the culture. For example, the expanded population of B cells is at least 10 times larger than the original isolated population of B cells. In another embodiment, the expanded population of B cells is at least 100 times larger than the original isolated population of B cells. In one embodiment, the expanded population of B cells is at least 500 times larger than the original isolated population of B cells.

Modification of B cells

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

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

[0092] Exemplary methods for transfecting B cells are provided in WO 2014/152832 and WO 2016/100932, both of which are herein incorporated by reference in their entireties. Transfection of B cells can be accomplished using any of a variety of methods available in the art for introducing DNA or RNA into a B cell. Suitable methods can include transfection using calcium phosphate, DEAE-dextran, electroporation, pressure-mediated transfection or "cell squeezing" (e.g., using the CellSqueeze microfluidic system, SQZ Biotechnologies), nanoparticle-mediated or liposome-mediated transfection, and transduction using a retrovirus or another virus, 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 5124259; US 5297983; US 5283185; US 5661018; US 6878548; US 7799555; US 8551780; and US 8633029. One example of a commercially available electroporation method suitable for B cells is the Nucleofector™ transfection technology.

[0093] Transfection can be performed prior to or during in vitro culturing of the isolated B cells in the presence of one or more of the activation and/or differentiation factors described above. 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 culturing. In one embodiment, the cells are transfected on day 1, 2, or 3 of in vitro culturing. In a specific embodiment, the cells are transfected on day 2. For example, the cells are electroporated on day 2 of in vitro culture to deliver, for example, 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 modified (e.g., by targeted integration of a follistatin transgene) prior to activation. In another embodiment, the cells are transfected or otherwise modified (e.g., by targeted integration of a follistatin transgene) during activation. In one embodiment, the cells are transfected or otherwise modified (e.g., by targeted integration of a follistatin transgene) after activation. In one embodiment, the cells are transfected or otherwise modified (e.g., by targeted integration of a follistatin transgene) prior to differentiation. In another embodiment, the cells are transfected or otherwise modified (e.g., by targeted integration of a follistatin transgene) during differentiation. In one embodiment, the cells are transfected or otherwise modified (e.g., by targeted integration of a follistatin transgene) after differentiation.

[0095] In one embodiment, a non-viral vector is used to deliver DNA or RNA (e.g., DNA or RNA comprising a sequence encoding a follistatin polypeptide) into 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 another transposon system 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 virus-independent vector systems can also be delivered via a viral vector known in the art or described below. For example, in some embodiments, a viral vector (e.g., a retrovirus, lentivirus, adenovirus, adeno-associated virus) is used to deliver one or more non-viral vectors (e.g., such as one or more of the aforementioned zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), clustered regularly interspaced short palindromic repeats (CRISPR), meganucleases, or any other enzymes/complementary vectors, polynucleotides, and/or polypeptides) capable of facilitating targeted integration. Accordingly, in some embodiments, a cell (e.g., B cells, such as memory B cells and/or plasma cells) can be modified to express an exogenous sequence (e.g., a sequence encoding a follistatin polypeptide) by a targeted integration method. Such methods are known in the art and can include cleaving an endogenous locus in a cell using one or more nucleases (e.g., ZFN, TALEN, CRISPR/Cas, meganuclease) and introducing a follistatin transgene into the cell such that it integrates into the endogenous locus and is expressed in the cell. The follistatin transgene can be contained in a donor sequence integrated into the host cell DNA at or near the point of nuclease cleavage.

[0097] Integration of an exogenous sequence (e.g., a sequence encoding a follistatin polypeptide) may occur through recombination. As will be understood by one of skill in the art, "recombination" refers to the process of exchanging genetic information between two polynucleotides, including, but not limited to, donor capture via non-homologous end joining (NHEJ) and homologous recombination. The recombination may be homologous recombination. For purposes of the present disclosure, "homologous recombination (HR)" refers to a specialized form of such exchange that occurs, for example, through the repair of double-strand breaks in cells via homology-directed repair mechanisms. This process utilizes nucleotide sequence homology whereby a "donor" molecule (e.g., a donor polynucleotide sequence or a donor vector comprising such a sequence) is used by the cellular DNA repair machinery as a template for repair of a "target" molecule (i.e., a molecule that has undergone a double-strand break), thereby transferring genetic information from the donor to the target. In some embodiments of HR-directed integration, the donor molecule may comprise at least 2 regions of homology to the genome ("homology arms"). In some embodiments, the homology arms may be, for example, at least 50-100 base pairs long. The homology arms may have sufficient DNA homology to a region of genomic DNA flanking the cleavage site where targeted integration can occur. Homology arms of the donor molecule may flank DNA (e.g., containing DNA encoding follistatin) to be integrated into the target genome or target DNA locus. Chromosome disruption followed by repair using the homologous region of plasmid DNA as a template may result in the transfer of an internal transgene flanked by 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 by up to 10 ⁵ -fold by deliberately creating a double-strand break near the target region (Hockemeyer et al. (2009) Nature Biotech. 27(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 a target cell (e.g., via recombination such as HR) can be used to modify a cell (e.g., a memory B cell or plasmablast) of the present invention.

[0099] A double-strand break (DSB) or nick can be generated by a site-specific nuclease such as zinc finger nuclease (ZFN), TAL effector domain nuclease (TALEN), meganuclease, or by using a CRISPR/Cas9 system such as crRNA/tract RNA (single guide RNA) to direct specific cleavage. See, e.g., Burgess (2013) Nature Reviews Genetics 14:80–81, Urnov et al. (2010) Nature 435(7042):646–51; U.S. Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060188987; 20090263900; 20090117617; 20100047805; 20110207221; 20110301073 and International Publication No. WO 2007/014275, the entire contents of which are incorporated by reference for all purposes.

[00100] In some embodiments, a cell (e.g., a memory B cell or plasmablast) is modified using zinc finger nuclease-mediated targeted integration of a donor construct (e.g., a follistatin donor construct). A zinc finger nuclease (ZFN) is an enzyme capable of recognizing and cleaving a target nucleotide sequence with specificity due to the association of a "zinc finger DNA binding protein" (ZFP) (or binding domain) that binds DNA in a sequence-specific manner via one or more zinc fingers, and a nuclease enzyme. ZFNs can contain any suitable cleavage domain (e.g., a nuclease enzyme) operably linked to the DNA binding domain of a ZFP to form an engineered ZFN that can facilitate site-specific cleavage of a target DNA sequence (see, e.g., Kim et al. (1996) Proc Natl Acad Sci USA 93(3):1156-1160). For example, ZFNs can contain a target-specific ZFP linked to the FOK1 enzyme or a portion of the FOK1 enzyme. In some embodiments, ZFNs used in a ZFN-mediated targeted integration method utilize two separate molecules, each comprising a FOK1 enzyme subunit, wherein each is linked to a ZFP, each ZFP has specificity for a DNA sequence flanking a cleavage target site, and when the two ZFPs bind to their respective DNA target sites, the FOK1 enzyme subunits approach each other and bind together, activating nuclease activity that cleaves the cleavage target site. ZFNs have been used to modify the genome in a variety of organisms (e.g., U.S. Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060188987; 20060063231; and International Publication No. WO 07/014,275, the entire contents of which are herein incorporated by reference). Custom-designed ZFPs and ZFNs are commercially available, e.g., from Sigma Aldrich (St. Louis, MO), and any location on DNA can be routinely targeted and cleaved using such custom-designed ZFNs.

[00101] In some embodiments, a cell (e.g., a memory B cell or plasmablast) is modified by CRISPR/Cas nuclease (e.g., CRISPR Cas9)-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 enters a bacterium, fragments of the invading DNA are translated into CRISPR RNA (crRNA) by the "immune" response. This crRNA then binds, through a region of partial complementarity, to another type of RNA called tracrRNA to guide the Cas9 nuclease to a region homologous to the crRNA in the target DNA called the "protospacer." Cas9 cleaves the DNA to produce blunt ends at DSBs at sites defined by a 20-nucleotide guide sequence contained within the crRNA transcript. Cas9 requires both crRNA and tracrRNA to site-specifically recognize and cleave the DNA. Currently, this system is designed such that crRNA and tracrRNA can be combined in a single molecule (a "single guide RNA"), and the equivalent crRNA portion of the single guide RNA can be designed to direct the Cas9 nuclease to a target of any desired sequence (see Jinek et al (2012) Science 337, pp. 816-821, Jinek et al, (2013), eLife 2:e00471, and David Segal, (2013) eLife 2:e00563). Thus, the CRISPR/Cas system can be designed to produce a DSB at a desired target in the genome, and DSB repair can be influenced by the use of repair inhibitors to enhance error-prone repair. As will be appreciated by one of skill in the art, other CRISPR nucleases in addition to Cas9 are known and suitable for use in the present invention.

[00102] In some embodiments, CRISPR/Cas nuclease-mediated integration utilizes type II CRISPR. Type II CRISPR is one of the best characterized systems and implements targeted double-strand DNA breaks in four sequential steps. First, two non-coding RNAs, a pre-crRNA array and tracrRNA, are transcribed from a CRISPR locus. Second, tracrRNA hybridizes to pre-crRNA repeat regions and mediates the processing of pre-crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to target DNA via Watson-Crick base pairing between a spacer in the crRNA and a protospacer in the target DNA following a protospacer adjacent motif (PAM), an additional requirement for target recognition. Fourth, Cas9 mediates cleavage of target DNA to form a double-strand break within the protospacer.

[00103] The Cas9-related CRISPR/Cas system comprises two non-coding RNA components: tracrRNA and a pre-crRNA array containing nuclease guide sequences (spacers) interspersed with identical direct repeats (DRs). In order for the CRISPR/Cas system to be used for genome engineering, both functions of these RNAs must be present (see Cong et al, (2013) Sciencexpress 1/10.1126/science 1231143). In some embodiments, tracrRNA and pre-crRNA are delivered via separate expression constructs or as separate RNAs. In other embodiments, a chimeric RNA is constructed wherein the engineered mature crRNA (providing target specificity) is fused to tracrRNA (providing interaction with Cas9) to produce a chimeric cr-RNA-tracrRNA hybrid (also referred to as a single guide RNA). (see Jinek, ibid., and Cong, ibid.).

[00104] In some embodiments, a single guide RNA containing both crRNA and tracrRNA can be engineered to direct the Cas9 nuclease to target any desired sequence (e.g., Jinek et al (2012) Science 337, p. 816-821, Jinek et al, (2013), eLife 2:e00471, David Segal, (2013) eLife 2:e00563). Thus, the CRISPR/Cas system can be engineered to generate a DSB at a desired target in the genome.

[00105] Custom-designed CRISPR/Cas systems are commercially available, such as from Dharmacon (Lafayette, CO), and any location on DNA can be routinely targeted and cleaved using such custom-designed single guide RNA sequences. Single-stranded DNA templates for recombination can be synthesized (e.g., by oligonucleotide synthesis methods known in the art and commercially available) or provided in a vector, such as a viral vector such as AAV.

[00106] In some embodiments, a cell (e.g., a memory B cell or plasmablast) is modified 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 comprising one or more TALE repeat domains/units. The repeat domains are involved in binding of the TALE to its cognate target DNA sequence. One "repeat unit" (also referred to as a "repeat") is typically 33-35 amino acids in length and has at least some sequence homology to other TALE repeat sequences in a native TALE protein. TAL effectors may comprise a nuclear localization sequence, an acidic transcriptional activation domain, and a centralized tandem repeat domain, wherein each repeat comprises approximately 34 amino acids, which are key to the DNA binding specificity of these proteins. (e.g. Schornack S, et al (2006) J Plant Physiol 163(3): 256-272). TAL effectors depend 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) Mol Gen Genet 218: 127-136). These DNA-binding repeats can be engineered into proteins with new combinations and numbers of repeats to produce artificial transcription factors that are able to interact with the new sequences and activate the expression of a non-endogenous reporter gene (e.g. Bonas et al (1989) Mol Gen Genet 218: 127-136). Engineered TAL proteins can be linked to the FokI cleavage half-domain to produce a TAL effector domain nuclease (TALEN) fusion protein to cleave a specific target DNA sequence (e.g., Christian et al (2010) Genetics epub 10.1534/genetics.110.120717).

[00107] Custom-designed TALENs are commercially available, for example from Thermo Fisher Scientific (Waltham, MA), and any location on DNA can be routinely targeted and cleaved.

[00108] In some embodiments, a cell (e.g., a memory B cell or plasmablast) is modified by meganuclease-mediated targeted integration of a donor construct (e.g., a follistatin donor construct). A meganuclease (or "homing endonuclease") is an endonuclease that binds and cleaves double-stranded DNA at a recognition sequence 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 cleavage sites of 15-40 base pairs and are generally grouped into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cyst box family, and the HNH family. Illustrative 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. Their 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.

[00109] In particular embodiments, the methods and compositions described herein utilize a nuclease comprising an engineered (non-natural) homing endonuclease (meganuclease). The recognition sequences of homing endonucleases and meganucleases, 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, 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 catalogs. In addition, the DNA binding specificity of homing endonucleases and meganucleases can be modified to bind non-natural target sites. See, e.g., 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 Publication No. 20070117128. The DNA binding domains of the homing and meganuclease endonucleases can be altered in the context of the nuclease as a whole (i.e., such that the nuclease includes a cognate cleavage domain) or can be fused to a heterologous cleavage domain. Custom-designed meganuclease is commercially available, for example, from New England Biolabs (Ipswich, MA), and any location on DNA can be routinely targeted and cleaved.

[00110] Modifying B cells may include introducing one or more nucleases (e.g., ZFN, TALEN, CRISPR/Cas, meganuclease) into a B cell, such as via one or more vectors encoding the nucleases, such that the vectors containing the encoded nucleases are taken up by the B cell. The vectors may be viral vectors.

[00111] In some embodiments, nucleases cleave a specific endogenous locus (e.g., a safe harbor gene or a locus of interest) in a cell (e.g., a memory B cell or a plasma cell), and introduce one or more exogenous (donor) sequences (e.g., transgenes) (e.g., one or more vectors containing the exogenous sequences). In such embodiments, the donor sequence can 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 a donor transgene (e.g., a follistatin donor transgene) can be accomplished through homologous repair (HDR), non-homologous repair mechanisms (e.g., NHEJ-mediated end joining), or insertions and/or deletions of nucleotides (e.g., endogenous sequence) at the site of integration of the transgene (e.g., a follistatin transgene) into the genome of the cell. In one embodiment, the method of transfecting a B cell comprises electroporating the B cell prior to contacting the B cell 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 to deliver the plasmid.

[00112] In one embodiment, the cells are transfected with a transposon. As used herein, the term "transposed" may, in some embodiments, refer to a cell that is transfected with a transposon. Numerous transposon systems are known in the art and are suitable for use in the present invention. For example, the sleeping beauty transposon system and the Piggybac transposon systems are well known in the art and are suitable for use in the present invention. See , e.g., Hackett PB, et al., Evaluating Risks 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, each of which is incorporated herein by reference in its entirety. In some embodiments, the cells are transfected with a Sleeping Beauty transposon. The Sleeping Beauty transposon may, in some embodiments, be a Sleeping Beauty T2 transposon or a Sleeping Beauty T4 transposon. In some embodiments, using the sleeping beauty transposon system may comprise transfecting (e.g., by electroporation) B cells 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, using the sleeping beauty transposon system may comprise transfecting (e.g., by electroporation) B cells 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 with a Piggybac transposon. In one embodiment, the cells are transfected with a transposon (e.g., a Sleeping beauty T2 or T4 transposon, or a Piggybac transposon) on a day ranging from day 1 to day 12 of in vitro culture. In one embodiment, the cells are transfected with a transposon (e.g., a Sleeping beauty T2 or T4 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 with a minicircle on a day ranging from day 1 to day 12 of in vitro culture. In one embodiment, cells are transfected using a minicircle on day 1, 2, 3, 4, 5, 6, 7, 8, or 9 of in vitro culture.

[00113] In one embodiment, the cells are transfected with a Sleeping Beauty transposon (e.g., a Sleeping beauty T2 or T4 transposon) on a day ranging from day 1 to day 12 of in vitro culture. In one embodiment, the cells are transduced with a sleeping beauty transposon system (e.g., a Sleeping beauty T2 or T4 transposon) on day 2 of in vitro culture via electroporation. In one embodiment, the cells are transduced with a sleeping beauty transposon system (e.g., a Sleeping beauty T2 or T4 transposon) on day 5 of in vitro culture via electroporation. In one embodiment, the cells are transduced with a sleeping beauty transposon system (e.g., a Sleeping beauty T2 or T4 transposon) on day 8 of in vitro culture via electroporation. In one embodiment, the cells are transduced using the Sleeping Beauty transposon system (e.g., the Sleeping beauty T2 or T4 transposon) on day 11 of in vitro culture via electroporation. In one embodiment, the cells are transduced using the Sleeping Beauty transposon system (e.g., the Sleeping beauty T2 or T4 transposon) on day 14 of in vitro culture via electroporation.

[00114] In one embodiment, the cells are transfected with the Piggybac transposon on a day ranging from day 1 to day 12 of in vitro culture. In one embodiment, the cells are transduced with the Piggybac transposon on day 2 of in vitro culture via electroporation. In one embodiment, the cells are transduced with the Piggybac transposon on day 5 of in vitro culture via electroporation. In one embodiment, the cells are transduced with the Piggybac transposon on day 8 of in vitro culture via electroporation. In one embodiment, the cells are transduced with the Piggybac transposon on day 11 of in vitro culture via electroporation. In one embodiment, the cells are transduced with the Piggybac transposon on day 14 of in vitro culture via electroporation.

[00115] In one embodiment, the B cells are contacted with a vector comprising a nucleic acid of interest operably linked to a promoter under conditions that allow transfection of at least a portion of the B cells. In one embodiment, the B cells are contacted with a vector comprising a nucleic acid of interest operably linked to a promoter under conditions that allow transfection of at least 5% of the B cells. In a further embodiment, the B cells are contacted with the vector under conditions that allow transfection of 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 as described herein are transfected, in which case the cultured B cells are contacted with a vector as described herein under conditions that allow transfection of 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.

[00116] 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, retroviral vectors, retroviral-adenoviral vectors, and vectors derived from herpes simplex viruses (HSVs), including amplicon-based vectors, replication-defective HSV, and attenuated HSV (see, e.g., Krisky, Gene Ther. 5: 1517-30, 1998; Pfeifer, Annu. Rev. Genomics Hum. Genet. 2:177-211, 2001, all of which are herein incorporated by reference in their entirety).

[00117] 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 particular 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 lentivirus pseudotyped with measles virus on day 1 of in vitro culture.

[00118] In one embodiment, B cells are transduced with retroviral vectors using any of a variety of methods known in the art (see, e.g., Science 12 April 1996 272:263-267; Blood 2007, 99:2342-2350; Blood 2009, 1 13:1422-1431 ; Blood 2009 Oct 8; 1 14(15):3173-80; Blood. 2003;101(6):2167-2174; Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, NY(2009)). Further description of viral transduction of B cells can be found in WO 2011/085247 and WO 2014/152832, the entire contents of each of which are hereby incorporated by reference.

[00119] For example, PBMC, B or T lymphocytes from donors and other B cells from cancer cells such as B-CLL can be isolated and cultured in IMDM or RPMI 1640 medium (GibcoBRL Invitrogen, Auckland, New Zealand) or other suitable medium as described herein, either serum-free or supplemented with serum (e.g., 5-10% FCS, human AB serum and serum substitutes) and penicillin/streptomycin, and/or other suitable additives such as transferrin and/or insulin. In one embodiment, the cells are seeded at 1 x 10 ⁵ cells in 48-well plates and the concentrated vector is added at various doses that can be routinely optimized by one of skill in the art using routine methods. In one embodiment, B cells are transferred to a monolayer of MS5 cells 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 refreshed periodically as needed. As known to those skilled in the art, other suitable media and supplements can be used as desired.

[00120] Particular embodiments relate to the use of retroviral vectors or vectors derived from retroviruses. "Retroviruses" are enveloped RNA viruses that are capable of infecting animal cells and that use the enzyme reverse transcriptase in the early stages of infection to produce a DNA copy of their RNA genome, which is then typically integrated into the host genome. Examples of retroviral vectors are Moloney murine leukemia virus (MLV)-derived vectors, murine stem cell virus-derived retroviral vectors that provide long-term stable expression in target cells such as hematopoietic progenitor 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 complex retrovirus-derived vectors such as lentiviral vectors.

[00121] In one embodiment, the B cells are contacted with a retroviral vector comprising a nucleic acid of interest operably linked to a promoter under conditions that allow transduction of at least a portion of the B cells. In one embodiment, the B cells are contacted with a retroviral vector comprising a nucleic acid of interest operably linked to a promoter under conditions that allow transduction of at least 2% of the B cells. In a further embodiment, the B cells are contacted with the vector under conditions that allow transduction of 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 resting B cells. In one specific embodiment, differentiated and activated B cells cultured in vitro as described herein are transduced, in which case the cultured differentiated/activated B cells are contacted with a vector as described herein under conditions that allow transduction of 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.

[00122] In particular embodiments, prior to transduction, the cells are pre-stimulated with Staphylococcus Aureus Cowan (SAC; Calbiochem, San Diego, CA) and/or IL-2 at appropriate concentrations known to those skilled in the art and optimized in a conventional manner. Other B cell activating factors (e.g., PMA) may be used, as known to those skilled in the art and described herein.

[00123] As noted above, in particular embodiments, lentiviral vectors are used. The term "lentivirus" refers to a genus of complex retroviruses capable of infecting both dividing and non-dividing cells. Examples of lentiviruses include HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2), visna-maedi virus, caprine arthritis-encephalitis virus, equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV). Lentiviral vectors may 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 95:1 1939-1 1944, 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, all of which are hereby incorporated by reference in their entirety).

[00124] It has been shown that resting T and B cells can be transduced with VSVG-coated LV carrying most of the HIV accessory proteins (vif, vpr, vpu, and nef) (see, e.g., Frecha et al., 2010 Mol. Therapy 18:1748). In particular embodiments, the retroviral vector comprises specific minimal sequences from a lentiviral genome, such as an HIV genome or an SIV genome. The lentiviral genome is typically organized into a 5' long terminal repeat (LTR) region, a gag gene, a pol gene, an 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 polyadenylation signals, and the R region separates the U3 and U5 regions. Transcribed R region sequences can occur at both the 5' and 3' ends of the viral RNA (see, e.g., RNA Viruses: A Practical Approach (Alan J. Cann, Ed., Oxford University Press, 2000); O Narayan, J. Gen. Virology. 70:1617-1639, 1989; Fields et al., Fundamental Virology Raven Press., 1990; Miyoshi et al., J Virol. 72:8150-7, 1998; and U.S. Patent No. 6,013,516, the entire disclosures of each of which are hereby incorporated by reference). Lentiviral vectors may contain any one or more of these lentiviral genomic elements, to regulate the activity of the vector as desired, or they may contain deletions, insertions, substitutions, or mutations in one or more of these elements, for example, to reduce the pathological effects of lentiviral replication, or to limit the lentiviral vector to a single cycle of infection.

[00125] Typically, a minimal retroviral vector comprises specific 5'-LTR and 3'-LTR sequences, one or more genes of interest (to be expressed in the target cell), one or more promoters, and a cis-activating sequence for RNA packaging. Other regulatory sequences may be included as described herein and as known in the art. The viral vector is typically cloned into a plasmid that can be transfected into a packaging cell line, such as a eukaryotic cell (e.g., 293-HEK), and also typically contains sequences that can be used to replicate the plasmid in bacteria.

[00126] In particular embodiments, the viral vector comprises sequences from the 5' and/or 3' LTR of a retrovirus, such as a lentivirus. The LTR sequences may be LTR sequences from any lentivirus from any species. For example, they may be LTR sequences from HIV, SIV, FIV, or BIV. Preferably, the LTR sequences are HIV LTR sequences.

[00127] In specific embodiments, the viral vector comprises the R and U5 sequences from a 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) that contains a mutation, substitution, or deletion that prevents the LTR sequences from controlling the expression of a downstream gene. A copy of the U3 region of the 3' LTR acts as a template for the production 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 prevented. 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, all of which are herein incorporated by reference in their entireties. Self-inactivating 3' LTRs can be produced by any method known in the art. In specific embodiments, the U3 element of the 3' LTR comprises a deletion of an enhancer sequence, preferably the TATA box, Spl and/or NF-kappa B regions. As a result of self-inactivation of the 3' LTR, the provirus integrated into the host cell genome comprises an inactivated 5' LTR.

[00128] The vectors provided herein typically comprise a gene that encodes a protein, such as follistatin, that is desirably expressed in one or more target cells. However, the vectors provided herein may also comprise genes that encode other molecules (such as, for example, siRNAs) that are desirably 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, such as transcriptional regulatory sequences such as promoters and/or enhancers, to regulate the expression of the gene of interest (e.g., follistatin) in a specific manner upon introduction of the gene into the target cell. In particular embodiments, transcriptional regulatory sequences that can be used are sequences that are highly subject to regulation with respect to activity, both temporally and spatially.

[00129] In particular embodiments, one or more additional genes can be introduced as a safety measure, such as to allow selective killing or depletion of a target cell within a heterogeneous population, such as within a human patient. In one non-limiting illustrative embodiment, the gene is a thymidine kinase (TK) gene, the expression of which sensitizes the target cell to the drug ganciclovir. In some embodiments, the additional gene is a cell surface protein tag gene. In some embodiments, the gene is a suicide gene. In some embodiments, the suicide gene is a caspase 9 suicide gene activated by a dimerizing drug (see, e.g., Tey et al., Biology of Blood and Marrow Transplantation 13:913-924, 2007).

[00130] In particular embodiments, one or more additional genes encoding a marker protein can be placed before or after a primary gene (e.g., a follistatin gene) in a viral or non-viral vector to allow identification and/or selection of cells expressing a desired protein (e.g., follistatin). Particular embodiments include an additional cell surface protein that can facilitate identification and/or selection of cells expressing a desired protein (e.g., follistatin). Particular embodiments include a fluorescent marker protein, such as green fluorescent protein (GFP) or red fluorescent protein (RFP), along with a primary gene of interest (e.g., a follistatin gene). If one or more additional reporter genes are included, IRES sequences or 2A elements separating the primary gene of interest (e.g., the follistatin gene) from the reporter gene and/or any other gene of interest may also be included.

[00131] In particular embodiments, genes encoding one or more selectable markers can be used. Examples include selectable markers that are effective in a eukaryotic cell or in a prokaryotic cell, such as a drug resistance gene encoding a factor necessary for the survival or growth of transformed host cells grown in a selective culture medium. Exemplary selectable genes encode proteins that provide resistance to antibiotics or other toxins, such as G418, hygromycin B, puromycin, zeocin, ouabain, blasticidin, ampicillin, neomycin, methotrexate, or tetracycline, complementing an auxotrophic deficiency, or the additive can be present on a separate plasmid and introduced by co-transfection with a viral vector. In one embodiment, the gene encodes a mutant dihydrofolate reductase (DHFR) that confers resistance to methotrexate. In other specific embodiments, genes encoding one or more cell surface receptors that can be used to label and detect or purify transfected cells (e.g., low affinity nerve growth factor receptor (LNGFR) or other such receptors that can be used as labeling systems for transduction can be used. See, e.g., Lauer et al ., Cancer Gene Ther. 2000 Mar;7(3):430-7.

[00132]IN In certain viral vectors, such as retroviral vectors, one or more heterologous promoters, enhancers, or both are used. In certain embodiments, the U3 sequence from the retroviral or lentiviral 5'-LTR can be replaced with a promoter or enhancer sequence in the viral construct. In certain embodiments, an "internal" promoter/enhancer is used that is located between the 5'-LTR and 3'-LTR sequences of the viral vector and is operably linked to a gene of interest (e.g., the follistatin gene).

[00133] "Functionally linked" and "functionally linked" mean, without limitation, that a gene (e.g., a follistatin gene) is in the correct location and orientation relative to a promoter and/or enhancer such that expression of the gene (e.g., a follistatin gene) can be induced when the promoter and/or enhancer are brought into contact with appropriate regulatory molecules. Any enhancer/promoter combination that either regulates (e.g., increases, decreases) expression of the viral RNA genome in the packaging cell line, or regulates expression of a selected gene of interest in an infected target cell, or both, can be used.

[00134] A promoter is an expression control element formed by a DNA sequence that allows polymerase binding and transcription to occur. Promoters are untranslated sequences located upstream (5') of the initiation codon of a selected gene of interest (usually within about 100 to 1000 bp) and control the transcription and translation of a coding polynucleotide sequence to which they are operably linked. Promoters may be inducible or constitutive. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as a change in temperature. Promoters may be unidirectional or bidirectional. Bidirectional promoters may be used to co-express two genes, for example, a gene of interest, such as follistatin, and a selectable marker. Alternatively, a bidirectional promoter configuration can be used, containing two promoters, each controlling the expression of a different gene, in opposite orientations in the same vector.

[00135] A variety of promoters are known in the art, as are methods for operably linking a promoter to a polynucleotide coding sequence. Both native promoter sequences and a variety of heterologous promoters can be used to control expression of a selected gene of interest. In particular embodiments, heterologous promoters are used because they typically allow stronger transcription and higher yields of the desired protein, compared to a native promoter.

[00136]IN In certain embodiments, heterologous viral promoters may be used. Examples of such promoters include promoters derived from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus, bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and simian virus 40 (SV40). In certain embodiments, a heterologous mammalian promoter may be used, such as an actin promoter, an immunoglobulin promoter, a heat shock promoter, or a promoter associated with the native sequence of a gene of interest (e.g., the follistatin gene). Typically, the promoter is compatible with a target cell, such as an activated B lymphocyte, a plasma B cell, a memory B cell, or other lymphocyte target cell.

[00137] In particular embodiments, one or more of RNA polymerase II and III promoters can be used. A 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, the entire contents of which are incorporated by reference. RNA polymerase II and III promoters also include any synthetic or engineered DNA fragments that can direct RNA polymerase II or III, respectively, to transcribe their downstream RNA coding sequences. Additionally, the RNA polymerase II or III (Pol II or III) promoter or promoters used as part of a viral vector can be inducible. Any suitable inducible Pol II or III promoter can be used in the methods described herein. Illustrative Pol II or III promoters include the tetracycline-responsive promoters described 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, all of which are hereby incorporated by reference in their entirety.

[00138] Non-limiting examples of constitutive promoters that can be used include the ubiquitin promoter, 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, the contents of each of which are hereby incorporated by reference). 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).

[00139] Additional examples of promoters include the ubiquitin-C promoter, the µ heavy chain promoter, or the human Ig heavy chain promoter (e.g., MH), and the κ light chain promoter or the human Ig light chain promoter (e.g., EEK), which are functional in B lymphocytes. The MH promoter comprises the human µ heavy chain promoter preceded by an iEµ enhancer flanked by matrix binding regions, and the EEK promoter comprises the κ light chain promoter preceded by an intronic enhancer (iEκ), a matrix binding 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). Accordingly, in particular embodiments, one or more of these promoter or enhancer elements may be used.

[00140] In one embodiment, one promoter drives expression of a selectable marker, and a second promoter drives expression of a gene of interest (e.g., a follistatin gene). For example, in one embodiment, the EF-1 alpha promoter drives production of a selectable marker (e.g., DHFR), and the miniature CAG promoter (see, e.g., Fan et al. Human Gene Therapy 10:2273-2285, 1999) drives expression of a gene of interest (e.g., follistatin).

[00141] As noted above, in particular embodiments, enhancer elements, such as an internal enhancer, are used to increase expression of a gene of interest. Enhancers are cis-activating DNA elements, typically about 10 to 300 bp in length, that act on a promoter to increase transcription from it. Enhancer sequences can be derived from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, insulin), such as the ιεµ enhancer, the ιεκ intronic enhancer, and the εκ 3' enhancer. Also included are enhancers from a eukaryotic virus, 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. The enhancers may be spliced into the vector at a position 5' or 3' of the antigen-specific polynucleotide sequence, but are preferably located in the region 5' of the promoter. One skilled in the art can select an appropriate enhancer based on the desired expression pattern.

[00142] In particular embodiments, promoters are selected to provide inducible expression of a gene of interest (e.g., a follistatin gene). A number of inducible expression systems are known in the art, including a tetracycline sensitive system and a lac repressor operator system. It is also envisaged that a combination of promoters can be used to obtain the desired expression of a gene of interest (e.g., a follistatin gene). One skilled in the art can select a promoter based on the desired expression pattern of the gene in the organism and/or target cell of interest.

[00143] Certain viral vectors contain cis-activating packaging sequences to facilitate the insertion of genomic viral RNA into the viral particle. Examples include psi sequences. Such cis-activating sequences are known in the art. In certain embodiments, the viral vectors described herein can express two or more genes, which can be accomplished, for example, by inserting an internal promoter that is operably linked to each individual gene after the first gene, by inserting an element that facilitates co-expression, such as an internal ribosome binding sequence (IRES) element (U.S. Patent No. 4,937,190, the disclosure of which is incorporated herein by reference) or a 2A element, or both. Simply by way of illustration, IRES or 2A elements 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 a heavy or light chain) can be located immediately downstream of a promoter, and a second coding region (encoding the other chain) can be located downstream of the first coding region, with an IRES or 2A element located between the first and second coding regions, preferably immediately preceding the second coding region. In other embodiments, the IRES or 2A element is used to co-express an unrelated gene, such as a reporter gene, a selectable marker, a cell surface protein, or a gene that enhances immune function. Examples of IRES sequences that can be used include, but are not limited to, 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 12:135-147, 1996; and Le et al., Nuc. Acids Res. 25:362-369, 1997, the entire contents of each of which are hereby incorporated by reference). One example of element 2A includes the F2A sequence from foot-and-mouth disease virus.

[00144] In particular embodiments, the vectors provided herein also contain additional genetic elements to achieve the desired result. For example, particular viral vectors may include a signal that facilitates nuclear entry of the viral genome in a target cell, such as the HIV-1 flap signal. As a further example, particular viral vectors may include elements that facilitate characterization of the proviral integration site in a target cell, such as an amber suppressor tRNA sequence. Particular viral vectors may contain one or more genetic elements engineered to enhance expression of a gene of interest (e.g., the follistatin gene). For example, a woodchuck hepatitis virus response element (WRE) can be included 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, all of which are hereby incorporated by reference in their entirety). As another example, a chicken β-globin insulator can also be included in the construct. This element has been shown to reduce the chance of integrated DNA being silenced in the target cell due to methylation and heterochromatinization effects. In addition, the insulator can protect an internal enhancer, a promoter, and an exogenous gene from the positive or negative effects of the position of the surrounding DNA at the site of integration on the chromosome. In particular embodiments, each of these genetic elements is used. In another embodiment, the viral vectors provided herein may also contain a universal chromatin uncoating element (UCOE) to enhance expression (see, e.g., Zhang F, et al., Molecular Therapy: The journal of the American Society of Gene Therapy 2010 Sep;18(9):1640-9).

[00145] In specific embodiments, the viral vectors (e.g., retroviral, lentiviral) described herein are "pseudotyped" using one or more selected viral glycoproteins or envelope proteins, generally to target selected cell types. Pseudotyping generally refers to the insertion of one or more heterologous viral glycoproteins onto the surface of a viral particle, often allowing the viral particle to infect a selected cell that is different from its normal target cells. The "heterologous" element is derived from a virus different from the virus from which the RNA genome of the viral vector is derived. Typically, the glycoprotein coding regions of the viral vector are genetically altered, such as by deletion, to prevent expression of its own glycoprotein. Simply as an illustration, the envelope glycoproteins gp41 and/or gp120 from an HIV-derived lentiviral vector are typically deleted prior to pseudotyping using a heterologous viral glycoprotein.

[00146] In specific embodiments, the viral vector is pseudotyped using a heterologous viral glycoprotein that targets B lymphocytes. In specific embodiments, the viral glycoprotein allows for selective infection or transduction of resting or non-dividing B lymphocytes. In specific embodiments, the viral glycoprotein allows for selective infection of plasma B lymphocytes, plasmablasts, and activated B cells. In specific embodiments, the viral glycoprotein allows for infection or transduction of resting B lymphocytes, plasmablasts, plasma cells, and activated B cells. In specific embodiments, the viral glycoprotein allows for infection of B-cell chronic lymphocytic leukemia cells. In one embodiment, the viral vector is pseudotyped using VSV-G. In another embodiment, the heterologous viral glycoprotein is derived from a measles virus glycoprotein, such as Edmonton measles virus. Particular pseudotyping embodiments include the measles virus glycoproteins hemagglutinin (H), 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, all of which are herein incorporated by reference in their entireties). In one embodiment, the viral vector is pseudotyped using gibbon leukemia virus (GALV). In one embodiment, the viral vector is pseudotyped using a feline endogenous retrovirus (RD114). In one embodiment, the viral vector is pseudotyped using a baboon endogenous retrovirus (BaEV). In one embodiment, the viral vector is pseudotyped using a murine leukemia virus (MLV). In one embodiment, the viral vector is pseudotyped using gibbon leukemia virus (GALV). In further embodiments, the viral vector contains an integrated antibody binding domain, such as one or more variable regions (e.g., heavy and light chain variable regions), that serve to target the vector to a particular cell type.

[00147] The preparation of viral vectors can be accomplished using any suitable genetic engineering techniques known in the art, including, but not limited to, standard restriction endonuclease digestion, ligation, transformation, plasmid purification, PCR amplification, and DNA sequencing techniques, such as those described 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)).

[00148] Any of a variety of methods known in the art can be used to produce useful retroviral particles whose genome contains an RNA copy of a viral vector. As one method, the viral vector can be introduced into a packaging cell line that packages the viral genomic RNA of the viral vector into viral particles with specificity for the desired target cell. The packaging cell line typically provides in trans the viral proteins necessary for packaging the viral genomic RNA into viral particles and infection of the target cell, including the gag structural proteins, the pol enzyme proteins, and the envelope glycoproteins.

[00149] In particular embodiments, the packaging cell line stably expresses particular essential or desirable viral proteins (e.g., gag, pol) (see, e.g., U.S. Patent No. 6,218,181, the disclosure of which is incorporated herein by reference). In particular embodiments, the packaging cell line is transiently transfected with plasmids encoding particular essential or desirable viral proteins (e.g., gag, pol, glycoprotein), including the measles virus glycoprotein sequences described herein. In one exemplary embodiment, the packaging cell line stably expresses gag and pol sequences, and the cell line is then transfected with a plasmid encoding a viral vector and a plasmid encoding a glycoprotein. Following introduction of the desired plasmids, the viral particles are collected and processed as appropriate, such as by ultracentrifugation, to produce a concentrated storage preparation of the viral particles. Illustrative 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.

Medicine

[00150] As used herein, a "gene of interest" or "gene" or "nucleic acid of interest" refers to a transgene to be expressed in a transfected target cell. In particular embodiments, the gene is the follistatin gene. While the term "gene" may be used, it does not imply that it is a gene as found in genomic DNA and is used interchangeably with the term "nucleic acid." In general, a nucleic acid of interest provides a nucleic acid suitable for encoding a drug (e.g., follistatin) and may comprise cDNA or DNA, and may or may not include introns, but typically does not include introns. As noted elsewhere herein, a nucleic acid of interest is operably linked to expression control sequences for efficient expression of the protein of interest in a target cell. In particular embodiments, the vectors described herein may contain one or more genes of interest, and may include 2, 3, 4, or 5 or more genes of interest, such as, for example, immunoglobulin heavy and light chain genes, which may be organized using an internal promoter as described herein.

[00151] The term "polynucleotide" or "nucleic acid" as used herein refers to mRNA, RNA, cRNA, cDNA or DNA. The term generally refers to a polymeric form of nucleotides at least 10 bases long, either ribonucleotides or deoxynucleotides, or a modified form of any type of nucleotide. The term includes single- and double-stranded forms of DNA and RNA. The nucleic acid or gene of interest may be any nucleic acid that encodes a protein of interest.

[00152] In some embodiments, in any of the embodiments described herein, a gene of interest, i.e., a follistatin protein gene, can be used. The follistatin protein can, in some embodiments, be any of the follistatin proteins set forth in SEQ ID NOs: 1-4. Thus, in some embodiments, a drug delivered to a genetically modified B cell as described herein can be a follistatin protein.

Follistatin

[00153] In various aspects, the present invention relates to B cells modified to express follistatin (e.g., one or more follistatin polypeptides). As used herein, the term "follistatin" refers to the follistatin (FST) protein family and follistatin-related proteins of any species. Follistatin is an autocrine glycoprotein expressed in nearly all tissues of higher animals. It was originally isolated from follicular fluid and identified as a protein fraction that inhibits follicle-stimulating hormone (FSH) secretion from the anterior pituitary gland, and was thus designated FSH suppressor protein (FSP). Its primary function was subsequently determined to be to bind to and neutralize members of the TGF-β superfamily, including, for example, activin, a paracrine hormone that enhances FSH secretion in the anterior pituitary gland.

[00154] In some embodiments, the terms "follistatin polypeptide" or "follistatin" are used to refer to polypeptides comprising any naturally occurring polypeptide of the follistatin family, as well as any variants thereof (including mutant, fragment, fusion, and peptidomimetic forms) that retain useful activity, including, for example, ligand binding (e.g., myostatin, GDF-11, activin A, activin B) or heparin binding. For example, in some embodiments, follistatin polypeptides can include polypeptides comprising an amino acid sequence derived from the sequence of any known follistatin having a sequence that is at least about 80% identical to the sequence of a follistatin polypeptide, and preferably at least 85%, 90%, 95%, 97%, 99%, or more identical.

[00155] Follistatin is a single-chain polypeptide with a molecular mass range of 31 to 49 kDa based on alternative mRNA splicing and variable protein glycosylation. The human gene encoding follistatin (FST) has six exons spanning 5,329 bp on chromosome 5q11.2 and produces two major transcripts: the FST344 variant transcript (1,122 bp) and the FST317 transcript (1,386 bp). Exon 1 of FST encodes the follistatin signal peptide, exon 2 encodes the N-terminal domain of follistatin, and exons 3-5 each encode a follistatin module. Due to alternative splicing, either exon 6A (encoding the acidic region in FST344) or exon 6B (containing two bases from the stop codon of FST317) are used (Shimasaki, S. et al., 1988).

[00156] These alternatively spliced mRNAs (FST344 and FST317) result in the production of 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, and follistatin 315 can be further proteolytically degraded to follistatin 303 (FST303). Amino acid sequence analysis revealed that the native human follistatin polypeptide contains five domains (from the N-terminal side): 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.

[00157] The 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) shown by single underlining, and the follistatin I-III domains (FSI, FSII, FSIII) shown by double underlining.

MVRARHQPGGLCLLLLLLCQFMEDRSAQA GNCWLRQAKNGRCQVLYKTEL SKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKET CENVDC GPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARC KEQPELEVQYQGRC KKT CRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPA SSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQC TGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEA ACSSGVLLEVKHSGSC N (SEQ ID NO: 1)

[00158] The processed (mature) variant of human follistatin (FST288) has the following amino acid sequence, with the N-terminal domain indicated by a single underline and the follistatin domains I-III indicated by double underlines. It is further understood that any of the initial G or N amino acids, prior to the first cysteine residue, may be removed by processing or intentionally removed without consequence, and polypeptides containing such slightly smaller polypeptides are further included.

GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWM

IFNGGAPNCIPCKET CENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKG

PVCGLDGKTYRNECALLKARCKEQPELEVQYQGRC KKT CRDVFCPGSSTC

VVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGR

SIGLAYEGKC IKAK SCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKS

DEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCN (SEQ ID NO: 2)

[00159] The human follistatin 315 (FST315) precursor (i.e., FST344) has the following amino acid sequence, with the signal peptide shown in bold, the N-terminal domain (FSN) shown by single underlining, and the follistatin I-III domains (FSI, FSII, FSIII) shown by double underlining (NCBI Accession No. AAH04107.1; 344 amino acids).

MVRARHQPGGLCLLLLLLCQFMEDRSAQA GNCWLRQAKNGRCQVLYKTEL

SKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKET CENVDC

GPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARC

KEQPELEVQYQGRC KKT CRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPA

SSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQC

TGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEA

ACSSGVLLEVKHSGSCN SISEDTEEEEDEDQDYSFPISSILEW

(SEQ ID NO:3)

[00160] Processed (mature) human FST315 has the following amino acid sequence, with the N-terminal domain indicated by a single underline and the follistatin I-III domains indicated by double underlines. It is further understood that any of the initial G or N amino acids, prior to the first cysteine residue, may be removed by processing or intentionally removed without consequence, and polypeptides containing such slightly smaller polypeptides are further included.

GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWM

IFNGGAPNCIPCKET CENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKG

PVCGLDGKTYRNECALLKARCKEQPELEVQYQGRC KKT CRDVFCPGSSTC

VVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGR

SIGLAYEGKC IKAK SCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKS

DEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSC NSISEDTEEEEED

EDQDYSFPISSILEW (SEQ ID NO: 4)

[00161]Polypeptides The follistatin proteins of the present invention may include any naturally occurring domain of the follistatin protein, as well as variants (e.g., mutant, fragment, and peptide mimetic forms) thereof that retain useful activity. For example, it is well known that FST315 and FST288 have high affinity for both activin (activin A and activin B) and myostatin (and the closely related GDF11), and follistatin domains (e.g., FSN and FSD I-III) are believed to be involved in binding such TGF-β ligands. However, it is believed that each of these three domains may have different affinities for these TGF-β ligands. For example, one study showed that polypeptide constructs containing only the N-terminal domain (FSN) and two FSDI domains in tandem retained high affinity for myostatin, had low or no affinity for activin, and stimulated systemic muscle growth when administered to mice via gene expression (Nakatani et al., The FASEB Journal, Vol. 22477-487 (2008)).

[00162] Accordingly, the present invention relates, in part, to follistatin protein variants that are shown to selectively bind and/or inhibit a given TGF-β ligand, compared to the native FST protein (e.g., maintaining high affinity for myostatin, while having significantly reduced affinity for activin).

[00163] Thus, the present invention provides polynucleotides (isolated or purified, or pure polynucleotides) encoding the therapeutics (e.g., follistatin) of the present invention for genetically modifying B cells, vectors (including cloning vectors and expression vectors) comprising such polynucleotides, and cells (e.g., host cells) transformed or transfected with a polynucleotide or vector of the present invention. In specific embodiments, in any of the embodiments described herein, a follistatin selected from the follistatin polypeptides of SEQ ID NOs: 1-4 can be used (e.g., for expression in a B cell). In specific embodiments, in any of the embodiments described herein, a follistatin that is a splice region variant of human follistatin FST344 can be used (e.g., for expression in a B cell). In particular embodiments, a polynucleotide (DNA or RNA) encoding a protein of interest (e.g., follistatin) of the present invention is provided. Expression cassettes encoding proteins of interest are also provided herein.

[00164] The present invention also relates to vectors comprising a polynucleotide of the present invention and, in particular, to recombinant expression constructs. In one embodiment, the present invention provides a vector comprising a polynucleotide encoding a protein of the present invention (e.g., follistatin), together with other polynucleotide sequences that cause or facilitate transcription, translation, and processing of such protein-encoding sequences. Suitable cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described, for example, in Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989). Exemplary cloning/expression vectors include cloning vectors, shuttle vectors, and expression constructs that may be based on plasmids, phagemids, phasmids, cosmids, viruses, artificial chromosomes, or any carrier nucleic acid known in the art that is suitable for amplifying, transferring, and/or expressing the polynucleotide contained therein.

[00165] As used herein, unless otherwise described with respect to viral vectors, a "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Exemplary vectors include plasmids, minicircles, transposons (e.g., the Sleeping Beauty transposon), yeast artificial chromosomes, self-replicating RNAs, and viral genomes. Certain vectors can replicate autonomously within a host cell, while other vectors can integrate into the host cell's genome and thus replicate 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 an expression control sequence and are thus capable of directing the expression of these sequences. In particular embodiments, the expression constructs are derived from plasmid vectors. Exemplary constructs include a modified pNASS vector (Clontech, Palo Alto, Calif.) having nucleic acid sequences encoding an ampicillin resistance gene, a polyadenylation signal, and a T7 promoter region; pDEF38 and pNEF38 (CMC ICOS Biologies, Inc.) having a CHEF1 promoter; and pD18 (Lonza) having a 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, Calif.; Novagen, Madison, Wis.; Pharmacia, Piscataway, NJ).

[00166]You can receive useful constructs comprising a dihydrofolate reductase (DHFR) coding sequence under suitable regulatory control to promote increased levels of fusion protein production, wherein such levels result from gene amplification following administration of an appropriate selective agent (e.g., methotrexate). In one embodiment, using a bifunctional transposon encoding a therapeutic gene (e.g., FST), together with drug resistance-DHFR, in combination with methotrexate (MTX) incubation to enrich for successfully transposed B cells, a more active product is obtained.

[00167] In general, recombinant expression vectors may include origins of replication and selectable markers that allow transformation of the host cell, and a promoter derived from a highly expressed gene to direct transcription of the downstream structural sequence, as described above. A vector in operable linkage with a polynucleotide of the present invention forms a cloning or expression construct. Exemplary cloning/expression constructs comprise at least one expression control element, such as a promoter, operably linked to a polynucleotide of the present invention. Additional expression control elements, such as enhancers, factor-specific binding sites, terminators, and ribosome binding sites, are also provided in the vectors and cloning/expression constructs of the present invention. The heterologous structural sequence of a polynucleotide of the present invention is assembled in the appropriate phase with translation initiation and termination sequences. Thus, for example, encoding nucleic acids as described herein can be included in any of a variety of expression vector constructs (e.g., minicircles) as a recombinant expression construct for expressing such protein in a host cell.

[00168] The appropriate DNA sequence(s) can be inserted into the vector, for example, by a variety of methods. Typically, the DNA sequence is inserted into appropriate restriction endonuclease cleavage site(s) by methods known in the art. Standard methods for cloning, isolating, amplifying, and purifying DNA are provided for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases, and the like, and various separation methods. A number of standard methods are described in, for example, Ausubel et al. (Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., Boston, MA, 1993); Sambrook et al. (Molecular Cloning, Second Ed., Cold Spring Harbor Laboratory, Plainview, NY, 1989); Maniatis et al. (Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, NY, 1982); Glover (Ed.) (DNA Cloning Vol. I and II, IRL Press, Oxford, UK, 1985); Hames and Higgins (Eds.) (Nucleic Acid Hybridization, IRL Press, Oxford, UK, 1985); and in other sources.

[00169] 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 regulable promoter) to direct the synthesis of mRNA. Representative examples of such expression control sequences include promoters from 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 a selectable marker. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase promoter, SV40 early and late, retrovirus LTR, and mouse metallothionein-l promoter. The selection of a suitable vector and promoter is well within the skill of the art, and the preparation of certain particularly preferred recombinant expression constructs comprising at least one promoter or regulatable promoter operably linked to a nucleic acid encoding a protein or polypeptide of the present invention are described herein.

[00170] Variants of the polynucleotides of the present invention are also provided. The 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 a defined sequence as described herein, or hybridize to one of these polynucleotides of a defined sequence under stringent hybridization conditions in 0.015 M sodium chloride, 0.0015 M sodium citrate at about 65-68°C, or in 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at about 42°C. Polynucleotide variants retain the ability to encode a binding domain or a fusion protein having the functionality described herein.

[00171] The term "stringent" is used to refer to conditions generally accepted in the art as stringent. The stringency of hybridization is principally 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 approximately 65-68°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at approximately 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, higher concentration of formamide or other denaturing agent) can also be used; however, this affects the percentage of hybridization. In cases where deoxyoligonucleotide hybridization is considered, additional illustrative stringent hybridization conditions include washing in 6xSSC, 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).

[00172] In a further aspect, the present invention provides a host cell transformed or transfected with or otherwise comprising any of the polynucleotides or vectors/expression constructs of the present invention (e.g., follistatin polynucleotides or vector/expression constructs). The polynucleotides or cloning/expression constructs of the present invention are introduced into suitable cells using any method known in the art, including transformation, transfection, and transduction (e.g., any of the methods described herein). Host cells include cells of a subject undergoing ex vivo cell therapy, including, for example, ex vivo gene therapy. Eukaryotic host cells are contemplated as an aspect of the present invention when they carry a polynucleotide, vector, or protein of the present invention, including, in addition to a 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 capable of modifying the glycosylation pattern of expressed multivalent binding molecules, see U.S. Patent Application Publication No. 2003/01 15614), 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 a eukaryotic cell known in the art to be used to express, and optionally isolate, a protein or peptide of the present invention. Also contemplated are prokaryotic cells, including Escherichia coli , Bacillus subtilis , Salmonella typhimurium , Streptomycete , or any prokaryotic cell known in the art to be suitable for expressing, and optionally isolating, a protein or peptide of the present invention. When isolating a protein or peptide from prokaryotic cells, it is particularly envisaged that methods known in the art for extracting protein from inclusion bodies can be used. The selection of an appropriate host as described herein is within the skill of the art. Host cells that glycosylate the fusion proteins of the present invention are contemplated.

[00173] The term "recombinant host cell" (or simply "host cell") refers to a cell containing a recombinant expression vector. It is to be understood that such terms are intended to refer not only to the particular cell in question, but also to the progeny of such cell. Since particular modifications may occur in subsequent generations, either due to mutation or environmental influences, such progeny may not, in fact, be identical to the original cell, but are still included within the scope of the term "host cell" as used herein. Recombinant host cells may be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying particular genes. Culture conditions for the particular host cells selected for expression, such as temperature, pH, etc., are readily apparent to those skilled in the art. Various mammalian cell culture systems may also be used to express the recombinant protein. Examples of mammalian expression systems include the COS-7 monkey kidney fibroblast lines described in Gluzman (1981) Cell 23:175 and other cell lines capable of expressing a compatible vector, such as the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors may contain an origin of replication, a suitable promoter and, optionally, an enhancer, as well as any necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, transcription termination sequences and 5' flanking non-transcribed sequences, such as described herein with respect to the production of multivalent binding protein expression constructs. DNA sequences derived from the SV40 splice and polyadenylation sites may be used to provide the necessary non-transcribed genetic elements. Introduction of the construct into the host cell can be accomplished by a variety of methods known 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).

Cells and compositions

[00174] In one embodiment, the modified B cells described herein are activated/differentiated in vitro and transfected to express a therapeutic agent as described herein (e.g., follistatin). In one embodiment, the modified B cells described herein are activated/differentiated in vitro and modified (e.g., using a targeted transgene integration method such as zinc finger nuclease, TALEN, meganuclease, or CRISPR/CAS9 transgene integration) to express a therapeutic agent as described herein (e.g., follistatin). In one embodiment, the compositions comprise B cells differentiated into plasma B cells, transfected or otherwise modified and expressing one or more proteins of interest (e.g., follistatin). Target cell populations, such as transfected or otherwise modified and activated B cell populations as described herein, can be administered either alone or in the form of a pharmaceutical composition in combination with diluents and/or other components such as cytokines or cell populations.

[00175] In one embodiment, modified B cells modified to express one or more proteins of interest (e.g., follistatin) are harvested from culture following in vitro activation/differentiation at a time point at which the modified B cells have optimal migratory capacity for a particular chemoattractant. In some embodiments, optimal migratory capacity may be present at day 7, day 8, or day 9 of B cell culture. In some embodiments, optimal migratory capacity may be present at day 5, day 6, or day 7 of B cell culture following transfection or modification. In some embodiments, the optimal migratory capacity may be present on day 8 of B cell culture following transfection or modification, or later in culture than day 8 following transfection or modification (e.g., day 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or later than day 20). In some embodiments, the optimal migratory capacity may be present before day 10 of B cell culture. In some embodiments, the optimal migratory capacity may be present before day 8 of B cell culture following transfection or modification. In some embodiments, the optimal migratory capacity may be present on day 6 or day 7 of B cell culture. In some embodiments, the optimal migratory capacity may be present on day 4 or day 5 of B cell culture following transfection or modification. In some embodiments, the optimal migratory capacity may be present up to day 9 of B cell culture. In some embodiments, the optimal migratory capacity may be present up to day 7 of B cell culture following transfection or modification. In some embodiments, the optimal migratory capacity is optimal for homing of the modified B cell to CXCL12. In some embodiments, the optimal migratory capacity is optimal for homing of the modified B cell to the bone marrow of a subject that has received one or more administrations of the modified B cells. In some embodiments, the B cells are harvested for administration to the subject at optimal capacity for migration to CXCL12 and/or into the subject's bone marrow at about day 7 to about day 9 in culture. In some embodiments, the B cells are harvested for administration to the subject at optimal capacity for migration to CXCL12 and/or into the subject's bone marrow at about day 5 to about day 7 in culture following transfection or modification. In some embodiments, the B cells are harvested for administration to the subject at an optimal capacity to migrate to CXCL12 and/or the bone marrow of the subject by about day 10 in culture. In some embodiments, the B cells are harvested for administration to the subject at an optimal capacity to migrate to CXCL12 and/or the bone marrow of the subject by about day 8 in culture following transfection or modification. In some embodiments, the optimal capacity to migrate is optimal for homing the modified B cell to CXCL13. In some embodiments, the optimal capacity to migrate is optimal for homing the modified B cell to a site of inflammation in a subject that has received one or more administrations of the modified B cells. In some embodiments, the B cells are harvested for administration to the subject at an optimal capacity to migrate to CXCL13 and/or the site of inflammation in the subject by about day 6 or about day 7 in culture. In some embodiments, the B cells are harvested for administration to the subject at an optimal capacity to migrate to CXCL13 and/or to the site of inflammation in the subject at about day 4 or about day 5 in culture following transfection or modification. In some embodiments, the B cells are harvested for administration to the subject at an optimal capacity to migrate to CXCL13 and/or to the site of inflammation by about day 10 in culture. In some embodiments, the B cells are harvested for administration to the subject at an optimal capacity to migrate to CXCL13 and/or to the site of inflammation by about day 8 in culture following transfection or modification.

[00176] In some embodiments, the optimal migratory capacity is optimal for homing of the modified B cell to both CXCL12 and CXCL13. In some embodiments, the B cells are harvested at optimal migratory capacity for homing to both CXCL12 and CXCL13 on day 7 of B cell culture. In some embodiments, the B cells are harvested at optimal migratory capacity for homing to both CXCL12 and CXCL13 on day 5 of B cell culture following transfection or modification.

[00177] In some embodiments, the modified B cells are collected when at least about 20% of the B cells migrate in a chemotaxis assay to a particular chemoattractant. For example, but not limited to this example, the modified B cells (e.g., producing FST) can be collected when at least about 20% of the B cells migrate in a chemotaxis assay to CXCL12. Or, in another non-limiting example, the modified B cells (e.g., producing FST) can be collected when at least about 20% of the B cells migrate in a chemotaxis assay to CXCL13. In addition, modified B cells (e.g., producing FST) can be collected when at least about 30% of the B cells migrate in a chemotaxis assay to a particular chemoattractant (e.g., CXCL12 or CXCL13), or when at least about 40%, 45%, 50%, 55%, 60%, 65%, or at least about 70% of the B cells migrate in a chemotaxis assay to a particular chemoattractant (e.g., CXCL12 or CXCL13). In addition, modified B cells (e.g., producing FST) can be collected when more than 70% of the B cells migrate in a chemotaxis assay. Such chemotaxis assays are known in the art.

[00178] Briefly, the cell compositions described herein may comprise a differentiated and activated B cell population that has been transfected and expresses a therapeutic agent as described herein (e.g., follistatin), in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline, lactated Ringer's solution, and the like; carbohydrates such as glucose, mannose, sucrose, or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions described herein are preferably formulated for intravenous or subcutaneous administration.

[00179] In one embodiment, the cell composition is assessed for purity prior to administration. In another embodiment, the cell composition is tested for drug production reliability. In one embodiment, the cell composition is tested for sterility. In another embodiment, the cell composition is screened to ensure that it matches the recipient subject.

[00180]In one embodiment, the modified B cell population is assessed for polyclonality prior to administration to a subject. In some embodiments, ensuring the polyclonal nature of the final cell product is an important safety parameter. Specifically, the emergence of a dominant clone may be considered as potentially contributing toin vivoin tumor formation or an autoimmune disease. Polyclonality can be assessed by any methods 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 modified B cell population. Because the B cell receptor undergoes changes during B cell development that make it unique between B cells, this method allows for a quantitative assessment of how many cells share the same B cell receptor sequence (meaning that they are clonal). Thus, in some embodiments, the more B cells in the modified B cell population that express the same B cell receptor sequence, the more clonal the population is and, thus, the less safe the population is for administration to a subject. In contrast, in some embodiments, the fewer B cells in a modified population of B cells that express the same B cell receptor sequence, the less clonal the population is (i.e., more polyclonal) and thus the population is safer to introduce into a subject.

[00181] In some embodiments, the modified B cells are administered to the subject after determining that they are sufficiently polyclonal. For example, the modified B cells can be administered to the subject after determining that no particular clone of B cells in the final population comprises more than about 0.2% of the total B cell population. The modified B cells can be administered to the subject after determining that no particular clone of B cells in the final population comprises 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 specific embodiments, modified B cells (e.g., producing (follistatin)) are administered to a subject after it has been determined that no particular B cell clone in the final population comprises more than about 0.03% of the total B cell population.

[00182] In one embodiment, the cell composition is stored and/or transported at 4°C. In another embodiment, the cell composition is frozen for storage and/or transport. The cell composition can be frozen, for example, at -20°C or -80°C. In one embodiment, the step of freezing the cell composition includes liquid nitrogen. In one embodiment, the cell composition is frozen using a controlled rate freezer. Accordingly, the methods described herein can further include a thawing step.

Methods of application

[00183] One aspect of the present invention relates to in vivo delivery of a drug (e.g., follistatin) by delivery of a modified B cell modified to express the drug (e.g., follistatin). In particular embodiments, B cells modified to express follistatin are used in methods of treating and/or preventing a chronic disease and disorder, and/or in methods of increasing muscle size or strength in a patient.

[00184] The modified B cells described herein can be administered in a manner suitable for the disease or disorder being treated or prevented.

[00185] The amount and frequency of administration may be determined by such factors as the condition of the patient and the type and severity of the patient's disease, although appropriate doses can be determined through clinical studies.

[00186] 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 sequentially administered to the subject. In one embodiment, three doses of modified B cells are sequentially administered to the subject. In one embodiment, a dose of modified B cells is administered weekly, biweekly, monthly, bimonthly, trimonthly, semiannually, annually, or biyearly to the subject. In one embodiment, a second or subsequent dose of modified B cells is administered to the subject when the amount of drug produced by the modified B cells decreases.

[00187] In one embodiment, a dose of modified B cells is administered to the subject at a specific frequency (e.g., once per week, once every two weeks, once per month, once every two months, or once every three months) until a desired amount (e.g., an effective amount) of the drug (e.g., follistatin) is detected in the subject. In one embodiment, the amount of the drug (e.g., follistatin) is monitored in the subject. In one embodiment, a subsequent dose of modified B cells is administered to the subject when the amount of the drug produced by the modified B cells decreases below the desired amount. In one embodiment, the desired amount is a range that produces the desired effect. For example, in a method of treating muscular dystrophy (e.g., Becker muscular dystrophy), the desired amount of follistatin can be the amount in the plasma of the subject to whom the modified B cells are administered. In some embodiments, the desired amount may be an amount of follistatin that results in a certain level of weight gain in the subject. In some embodiments, the desired amount may be an amount of follistatin that results in a certain level of strength gain in the subject. In some embodiments, the desired amount may be an amount of follistatin that results in a certain level of weight gain in the subject.

[00188] When an "effective amount" or "therapeutic amount" is indicated, the precise amount of the compositions described herein to be administered can be determined by the physician taking into account individual differences in the age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject). The B-cell compositions can also be administered multiple times at the appropriate dose(s). The cells can be administered using infusion techniques well known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).

[00189] The optimal dose and treatment regimen for a particular patient can be determined by a medical professional by monitoring the patient for signs of disease and adjusting the treatment accordingly. The treatment can also be adjusted by measuring the levels of the drug (e.g., follistatin) in a biological sample (e.g., a body fluid such as plasma, or a tissue sample), which can also be used to assess the effectiveness of the treatment, and the treatment can be adjusted upward or downward accordingly.

[00190] In some aspects of the present invention, an optimal dose of modified B cells for a multiple dose regimen may be determined by first determining an optimal concentration of B cells in a single dose for a subject, reducing the number of B cells present at the optimal concentration in a single dose to obtain a suboptimal concentration of modified B cells in a single dose, and administering two or more doses of the suboptimal concentration of modified B cells in a single dose to the subject. In some aspects, 2, 3, or more doses of the suboptimal concentration of modified B cells in a single dose are administered to the subject. In some aspects, administering 2, 3, or more doses of the suboptimal concentration of modified B cells in a single dose to the subject results in synergistic in vivo production of a therapeutic polypeptide that the modified B cells are modified to express. In some aspects, the suboptimal single dose concentration is 1/2 or 3, 4, 5, 6, 7, 8, 9, 10 or more times less than the optimal single dose concentration. In some aspects, the therapeutic polypeptide is follistatin.

[00191] In some aspects of the present invention, lower amounts of transfected B cells described herein can be administered, in the range of 10 ⁶ /kilogram (10 ⁶ -10 ¹¹ per patient). In particular embodiments, the B cells are administered at 1×10 ⁴ , 5×10 ⁴ , 1×10 ⁵ , 5×10 ⁵ , 1×10 ⁶ , 5×10 ⁶ , 1×10 ⁷ , 5×10 ⁷ , 1×10 ⁸ , 5×10 ⁸ , 5×10 ⁹ , 1×10 ¹⁰ , 5×10 ¹⁰ , 1×10 ¹¹ , 5×10 ¹¹ or 1×10 ¹² cells to a subject. The B cell compositions can be administered multiple times at doses in these ranges. The cells can be autologous or heterologous (e.g., allogeneic) to the patient undergoing therapy. If desired, the treatment can also include the 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.), as described herein, to enhance the induction of an immune response and the engraftment of the infused B cells.

[00192] Administration of the subject compositions can be by any convenient route, including aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intrathecally, intramuscularly, via intravenous (iv) injection, or intraperitoneally. The compositions described herein can be administered directly into the nervous system of a patient. In one embodiment, the B cell compositions described herein are administered to a patient via intradermal or subcutaneous injection. In another embodiment, the B cell compositions as described herein are preferably administered via iv injection. The B cell compositions can be injected directly into a tumor, lymph node, bone marrow, or site of infection.

[00193] In 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, polymeric materials can be used (see Medical Applications of Controlled Release, 1974, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla.; Controlled Drug 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 another embodiment, the controlled release system can be placed in proximity to the therapeutic target, such that only a fraction of the systemic dose is required (see, e.g., Medical Applications of Controlled Release, 1984, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla., vol. 2, pp. 1 15-138).

[00194] The B cell compositions described herein may also be administered using any number of matrices. Matrices have been used for several years in the context of tissue engineering (see, e.g., Principles of Tissue Engineering (Lanza, Langer, and Chick (eds.)), 1997). The present invention utilizes such matrices in the novel context of acting as an artificial lymphoid organ for the maintenance and preservation of B cells. Accordingly, the present invention can employ those matrix compositions and formulations that have been shown to be useful in tissue engineering. Accordingly, the type of matrix that can be used in the compositions, devices, and methods of the present invention is virtually unlimited and can include both biological and synthetic matrices. In one specific example, the 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 matrices have features commonly associated with biocompatibility when administered to a mammalian host. Matrices may be formed from natural and/or synthetic materials. Matrices may be non-biodegradable, in cases where it is desirable to leave permanent structures or retrievable structures in the animal's body, such as an implant; or biodegradable. Matrices may be in the form of sponges, implants, tubes, telfa plugs, fibers, hollow fibers, lyophilized components, gels, powders, porous compositions, or nanoparticles. In addition, matrices can be designed to allow for the sustained release of seeded cells or produced cytokine or other active substance. In particular embodiments, a matrix according to the present disclosure is flexible and elastic, and can be described as a semi-solid scaffold permeable to substances such as inorganic salts, aqueous liquids, and dissolved gaseous agents, including oxygen.

[00195] Matrix is used in the present description as an example of a biocompatible substance. However, the present description is not limited to matrices and thus, whenever the term matrix or matrices appear, these terms should be understood to include devices and other substances that allow the retention of cells or the passage of cells, are biocompatible and are either capable of allowing the passage of macromolecules directly through the substance such that the substance itself is a semipermeable membrane, or are used in combination with a specific semipermeable substance.

[00196] In specific embodiments of the present invention, B cells transfected and activated using the methods described herein or other methods known in the art are administered to a patient in combination with (e.g., prior to, concurrently with, or subsequent to) any number of appropriate therapeutic options, including, but not limited to, treatments using agents such as antiviral agents, chemotherapy, radiation, immunosuppressive agents such as cyclosporine, bisulfine, bortezomib, azathioprine, methotrexate, mycophenolate, and FK506, antibodies or other immunodestructive agents such as campath, anti-CD3 antibodies, or other therapeutic antibodies, cytoxin, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation. These drugs either inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506), the proteasome (bortezomib), or inhibit the p70S6 kinase, which is important for growth factor-induced signaling (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 (supra)). In a further embodiment, the cell compositions disclosed herein are administered to a patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) bone marrow transplantation, T cell depleting therapy using either chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or Campath. In one embodiment, the cell compositions disclosed herein are administered following B cell depleting therapy such as CD20 reactive agents, such as Rituxan®. In one embodiment, the cell compositions disclosed herein are administered following B cell depleting therapy using an agent such as bortezomib. For example, in one embodiment, subjects can be treated with standard treatment using high dose chemotherapy followed by peripheral blood stem cell transplantation. In particular embodiments, following transplantation, subjects are infused with expanded immune cells disclosed herein. In a further embodiment, the expanded cells are administered before or after surgery.

[00197] The dose of the above drugs to be administered to a patient may be varied depending on the exact nature of the condition being treated and the recipient of the treatment. Dose scaling for administration to humans may be accomplished in accordance with accepted practice in the art.

[00198] Modified B cells can be used to treat or prevent various diseases and disorders. In particular embodiments, B cells modified to express follistatin are used in methods for increasing muscle size or strength in a patient. For example, the present invention provides a method for increasing muscle size or strength in a subject, comprising administering an effective amount of a B cell expressing a follistatin polypeptide. The increased muscle size or strength can occur generally in the subject or can occur in a target muscle. The target muscle may be destroyed, weakened, or failed, as may occur in muscle disorders including muscular dystrophies (such as Duchenne muscular dystrophy, Becker muscular dystrophy, Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, rigid spine syndrome, Ullrich syndrome, Fukuyama muscular dystrophy, Walker-Warburg syndrome, myo-oculo-brain disease, facioscapulohumeral muscular dystrophy), congenital muscular dystrophy, myotonic dystrophy (Steiner disease), non-dystrophic myotonia, spinal muscular atrophy with periodic paralysis, familial amyotrophic lateral sclerosis, hereditary motor and sensory neuropathy, Charcot-Marie-Tooth disease, chronic inflammatory neuropathy, distal myopathy, myotubular/centronuclear myopathy, nemaline myopathy, small core myopathy, central core myopathy, desminopathy, inclusion body myositis, mitochondrial myopathy, congenital myasthenic syndrome, postpolio muscle dysfunction, and the disorders described in Emery (2002) The Lancet, 359:687-695; and Khurana et al. (2003) Nat. Rev. Drug Disc., 2:379-386), inflammatory muscle disorders (such as inclusion body myositis), muscle injury or trauma, muscle disuse (such as may occur after prolonged bed rest or immobilization of a limb), and muscle atrophy or wasting due to aging, cancer, or chronic diseases of various types. For example, in some cases, muscle may be damaged, weakened, or insufficiently strong due to sarcopenia. In some cases, muscle may be damaged, weakened, or insufficiently strong due to spinal muscular atrophy (SMA). In some cases, muscle may be damaged, weakened, or insufficiently strong due to amyotrophic lateral sclerosis (ALS). In some cases, muscle may be damaged, weakened, or insufficiently strong due to Pompe disease. The methods may also involve increasing the size or strength of muscle in a healthy muscle.

[00199] In some embodiments, the present invention provides a method of treating a disease or disorder in an individual comprising administering a B cell genetically modified to express follistatin (e.g., follistatin+ B cells expressing a variant FST344 transcript) to a subject in need thereof.

[00200] In some embodiments, the present invention relates to a method of treating a muscle disorder in an individual, comprising administering a B cell genetically modified to express follistatin (e.g., follistatin+ B cells expressing a transcript variant of FST344) to a subject having, or suspected of having, such a muscle disorder, wherein the muscle disorder is muscular dystrophy. In some embodiments, the muscular dystrophy is selected from Duchenne muscular dystrophy, Becker muscular dystrophy, Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, rigid spine syndrome, Ullrich syndrome, Fukuyama muscular dystrophy, Walker-Warburg syndrome, myo-oculo-brain disease, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, myotonic dystrophy (Steiner disease), non-dystrophic myotonia, spinal muscular atrophy with periodic paralysis, familial amyotrophic lateral sclerosis, hereditary motor and sensory neuropathy, Charcot-Marie-Tooth disease, chronic inflammatory neuropathy, distal myopathy, myotubular/centronuclear myopathy, nemaline myopathy, small core myopathy, central core myopathy, desminopathy, inclusion body myositis, mitochondrial myopathy, congenital myasthenic syndrome, postpolio muscle dysfunction, and the disorders described in Emery (2002) The Lancet, 359:687-695; and Khurana et al. (2003) Nat. Rev. Drug Disc, 2:379-386).

[00201] In some embodiments, the present invention relates to a method of treating a muscle disorder in an individual, comprising administering a B cell genetically modified to express follistatin (e.g., follistatin+ B cells expressing a variant FST344 transcript) to a subject having, or suspected of having, such a muscle disorder, wherein the muscle disorder is an inflammatory muscle disorder. In some embodiments, the inflammatory disorder is inclusion body myositis.

[00202] In some embodiments, the present invention relates to a method of treating a muscle disorder in an individual, comprising administering a B cell genetically modified to express follistatin (e.g., follistatin+ B cells expressing a variant FST344 transcript) to a subject having, or suspected of having, such a muscle disorder, wherein the muscle disorder is caused by injury or trauma to a muscle.

[00203] In some embodiments, the present invention relates to a method of treating a muscle disorder in an individual, comprising administering a B cell genetically modified to express follistatin (e.g., follistatin+ B cells expressing a variant FST344 transcript) to a subject having, or suspected of having, such a muscle disorder, wherein the muscle disorder is caused by muscle disuse (e.g., as may occur following prolonged bed rest or limb immobilization).

[00204] In some embodiments, the present invention relates to a method of treating a muscle disorder in an individual, comprising administering a B cell genetically modified to express follistatin (e.g., follistatin+ B cells expressing a variant FST344 transcript) to a subject having, or suspected of having, such a muscle disorder, wherein the muscle disorder is selected from muscle wasting or weakening due to aging, cancer, or chronic diseases of various types.

[00205] In some embodiments, the present invention relates to a method of treating an individual exhibiting mild, moderate, or severe muscle weakness, muscle wasting, and/or effects on independent locomotion, comprising administering a B cell genetically modified to express follistatin (e.g., follistatin+ B cells expressing a variant FST344 transcript) to the subject.

[00206] In some embodiments, the present invention relates to a method of treating an individual exhibiting mild, moderate, or severe muscle frailty, muscle hypertrophy, muscle pseudohypertrophy, joint contracture, skeletal deformity, cardiomyopathy, dysphagia, bowel and bladder dysfunction, muscle ischemia, cognitive impairment, behavioral dysfunction, social impairment, scoliosis, and/or respiratory impairment, comprising administering a B cell genetically modified to express follistatin (e.g., follistatin+ B cells expressing a variant FST344 transcript) to the subject.

[00207] In specific embodiments, the present invention relates to a method of treating muscular dystrophy in an individual, comprising administering a B cell genetically modified to express follistatin (e.g., follistatin+ B cells expressing a transcript variant of FST344) to a subject having, or suspected of having, Becker muscular dystrophy.

[00208] In some embodiments, a single, maximally effective dose of follistatin+ B cells (e.g., follistatin+ B cells expressing a variant FST344 transcript) is administered to the subject. In some embodiments, two or more doses of follistatin+ B cells (e.g., follistatin+ B cells expressing a variant FST344 transcript) are administered to the subject, thereby maximizing the number of engrafted follistatin+ B cells. In some embodiments, the two or more doses of follistatin+ B cells (e.g., follistatin+ B cells expressing a variant FST344 transcript) that are administered to the subject contain 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., follistatin+ B cells expressing a variant FST344 transcript) are administered to a subject at a dose of follistatin+ B cells that is below the maximally effective single dose of follistatin+ B cells, the result is a synergistic increase in follistatin production. In one embodiment, administration of follistatin+ B cells to a subject results in normal follistatin levels observed in a healthy, control subject. In one embodiment, administration of follistatin+ B cells to a subject results in greater than normal follistatin levels in the subject. In one embodiment, administration of follistatin+ B cells (e.g., follistatin+ B cells expressing a variant FST344 transcript) to a subject increases the subject's strength. In one embodiment, administration of follistatin+ B cells (e.g., follistatin+ B cells expressing a variant FST344 transcript) to a subject increases the subject's strength compared to a normal level. In one embodiment, administration of follistatin+ B cells (e.g., follistatin+ B cells expressing a variant FST344 transcript) to a subject prevents loss of strength in the subject.

EXAMPLES

EXAMPLE 1

OBTAINING FOLLISTATIN-EXPRESSING B CELLS

[00209]Received constructions transposonSleeping Beautyand transposases for transposition and expression of human follistatin (FST). Transposons were assembled to obtain FST gene integration and expression in B cells. We used the EEK promoter, consisting of promoter and enhancer elements from the human immunoglobulin gene, as well as other previously described regulatory elements, to achieve high-level expression in B cells. To test FST transposition and expression, human B cells were isolated from two separate donors and expanded in culture in B cell culture medium and incubated at 37°C in a 5% CO incubator.₂, were electroporated on day 3 with pKT2/EEK-FST344 plus mRNA encoding the SB100x transposase. Cell lysates obtained at 2, 5, 8, and 11 days post-electroporation (i.e., days 5, 7, 11, and 14 in culture) contained significantly more FST than untransfected wild-type cells, demonstrating the efficiency of the SB transposon system to achieve high levels of FST expression in expanded human B cells (FIG. 5A). Notably, FST expression persisted from days 7 to 14, after a slight decrease from day 5 (likely due to initial episomal expression of FST), suggesting stable integration of the construct into B cells. Indeed, RT-PCR confirmed stable integration of the FST insert into the B cell genome (FIG. 5B). These FST-expressing B cells are designated as FST+ B cells in the following examples.

EXAMPLE 2

FST IN VIVO PRODUCTS

[00210] To determine whether B cells modified according to the present invention are capable of increasing FST production in vivo , the inventors injected wild-type mice with FST+ B cells obtained in Example 1.

[00211] Specifically, four mice were injected intravenously (tail vein) on day 0 with vehicle (500 μL phosphate-buffered saline (PBS)) or 2× ¹⁰⁶ human FST+ B cells diluted in vehicle (PBS) to 500 μL. Additionally, the mice were infused intraperitoneally (ip) on day -7 with 3× ¹⁰⁶ primary autologous peripheral blood cells enriched for CD4+ T cells to provide support for B cells transposed with pKT2/EEK-FST plus mRNA encoding the SB100x transposase. We observed a two-fold increase in human FST levels in the plasma of mice treated with FST+ B cells, providing strong evidence of successful adoptive transfer of human B cells (Figure 1). FST levels peaked at approximately day 28 post-infusion and decreased to near-normal levels by day 35. Although not obtained in FST-deficient animals, the results of this experiment nonetheless provide an example of the human FST levels that can be achieved following administration of a highly active FST+ B cell population to wt mice. The present inventors found that plasma FST levels correlated with plasma human IgG levels; thus, providing evidence of adoptive transfer and engraftment of FST+ B cells (Figures 2A-2D).

[00212] To determine whether FST+ B cells had any effect on the treated mice, we monitored the weight of control and FST+ B cell-treated mice 35 days after infusion. As shown in Figure 3, FST+ B cell-treated mice grew an average of 4.1% (0.9 grams) more than vehicle control-treated mice. Additionally, the increase in weight gain corresponded to a significant increase in strength in the forepaw elevated bar walk test (16% improvement in FST+ B cell-treated mice compared to vehicle control) (FIG. 4A); the quadruped elevated bar walk test (23% improvement in FST+ B cell-treated mice compared to vehicle control) (FIG. 4B); and suspension test (23% improvement in FST+ B cell-treated mice compared to vehicle controls) (FIG. 4C).

[00213] Thus, these data demonstrate that B cells can be used in the methods described herein to express and deliver FST to a subject to induce mass gain and improve strength.

[00214] The various embodiments described above can be combined to provide additional embodiments. The entire contents of all U.S. Patents, U.S. Patent Application Publications, U.S. Patent Applications, foreign patents, foreign patent applications, and non-patent publications referenced in this specification and/or listed in the Application Data Sheet are incorporated herein by reference. Aspects of the embodiments can be modified if necessary to utilize the concepts of various patents, applications, and publications to provide still additional embodiments.

[00215] These and other changes may be made to the embodiments in light of the above detailed description. Generally, in the following claims, the terms used are not to be construed as limiting the claims to the specific embodiments described in the description and claims, but are to be construed as including all possible embodiments together with the full scope of equivalents permitted by such claims. Accordingly, the claims are not limited by the description.

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LIST OF SEQUENCES

<110> Immunsoft Corporation

Scholz, Matthew Rein

Herbig, Eric J.

McIvor, R. Scott

<120> B CELLS GENETICALLY MODIFIED TO SECRETE FOLLISTATIN,

AND METHODS OF THEIR USE FOR THE TREATMENT OF FOLLISTATIN-ASSOCIATED DISEASES,

CONDITIONS, DISORDERS AND TO INCREASE MUSCLE GROWTH AND STRENGTH

<130> IMCO-008/01WO 312423-2028

<150> US 62/644362

<151> 2018-03-16

<150> US 62/644356

<151> 2018-03-16

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<---

Claims (87)

1. A recombinant B cell for producing follistatin in a subject, comprising a follistatin gene, wherein the B cell has been transduced using a DNA construct encoding a transposon system and a DNA construct encoding a follistatin polypeptide. 2. The recombinant B cell of claim 1, wherein the follistatin gene is operatively linked to a promoter. 3. The recombinant B cell of claim 1 or 2, wherein the follistatin gene is a human follistatin gene. 4. The recombinant B cell of any one of claims 1 to 3, wherein the follistatin gene is a variant of the FST-344 splice site of human follistatin. 5. A recombinant B cell according to any one of claims 1 to 4, wherein the B cell is a human B cell. 6. The recombinant B cell of any one of claims 1-5, wherein the transposon system is a sleeping beauty transposon system or a piggybac transposon system. 7. A recombinant B cell according to any one of claims 1-6, wherein the transduction is carried out via a virus. 8. A recombinant B cell according to any one of claims 1 to 7, wherein the B cell comprises a follistatin gene because it has been transduced with a retrovirus, lentivirus, adenovirus or adeno-associated virus containing a follistatin gene. 9. A recombinant B cell for producing follistatin in a subject, comprising a follistatin gene, wherein the B cell is modified to comprise the follistatin gene using a targeted integration method. 10. The recombinant B cell of claim 9, wherein the targeted integration utilizes one or more zinc finger nucleases, transcription activator-like effector nucleases (TALENs) and/or CRISPR/Cas systems. 11. The recombinant B cell of claim 9, wherein the B cell is modified to contain a follistatin gene by introducing a follistatin encoding nucleic acid using a method selected from the group consisting of retroviral vectors, lentiviral vectors, adeno-associated viral vectors, adenoviral vectors, any other RNA or DNA viral vectors, non-viral DNA and/or RNA encoding follistatin introduced using chemical or physical methods such as lipofection, polycation complexation, electroporation. 12. The recombinant B cell of any one of claims 1-11, wherein the follistatin polypeptide is secreted by the recombinant B cell. 13. A method of delivering follistatin to a subject comprising administering a recombinant B cell according to any one of claims 1-12. 14. A method for delivering follistatin to a subject in need thereof, comprising administering a recombinant B cell according to any one of claims 1-12. 15. The method according to claim 13 or 14, wherein the subject is a mammal. 16. The method according to any one of paragraphs 13-15, wherein the subject is a human. 17. The method according to any one of paragraphs 13-16, wherein the subject has muscular dystrophy. 18. The method according to any one of paragraphs 13-17, wherein the subject has Becker muscular dystrophy. 19. The method of any one of claims 13-18, wherein administering the recombinant B cell to the subject provides treatment for the disease, disorder, or condition of the subject. 20. The method of any one of claims 13-18, wherein administering the recombinant B cell to the subject provides treatment for muscular dystrophy. 21. The method of any one of claims 13-20, wherein administering the recombinant B cell to the subject causes an increase in the subject's body weight. 22. The method of claim 21, wherein the subject's body weight increases by at least about 4%. 23. The method according to claim 22, wherein a significant increase in body weight occurs within 30 days. 24. The method of claim 22, wherein a significant increase in body weight occurs in approximately 30 days. 25. The method of any one of claims 13-24, wherein administering the recombinant B cell to the subject causes an increase in muscle mass in the subject. 26. The method of any one of claims 13-25, wherein administering the recombinant B cell increases muscle strength in the subject. 27. The method of any one of claims 13-26, wherein administration of the recombinant B cell results in an increase in follistatin levels in the plasma of the subject. 28. A method of treating or alleviating a muscle disorder by administering a recombinant B cell according to any one of claims 1-12, wherein the muscle disorder is characterized by a decrease in follistatin. 29. A method for preventing a muscle disorder by administering a recombinant B cell according to any one of claims 1-12, wherein the muscle disorder is characterized by a decrease in follistatin. 30. A method of treating or ameliorating muscular dystrophy by administering a recombinant B cell according to any one of claims 1-12. 31. A method for preventing muscular dystrophy by administering a recombinant B cell according to any one of claims 1-12. 32. The recombinant B cell of any one of claims 1-12, 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 claim 32, wherein the recombinant B cell is derived from a B cell precursor obtained from the subject. 34. The recombinant B cell of claim 32, wherein the recombinant B cell is derived from a cell obtained from a subject that has differentiated into a B cell or a B cell precursor. 35. The recombinant B cell of any one of claims 1-12 and 32-34, wherein the recombinant B cell is modified by (a) collecting and isolating immunocytes from the blood of a subject; (b) transduction of cells with DNA encoding follistatin; (c) ex vivo propagation of the selected cells; and (d) differentiation of expanded cells ex vivo into plasma cells and/or plasmablasts; 36. The recombinant B cell of claim 35, wherein the isolated immunocytes from step a are CD19 positive cells. 37. A recombinant B cell according to claim 35 or 36, wherein the transduction in step b is carried out by electroporation. 38. The recombinant B cell of any one of claims 35-37, wherein the differentiated cells are CD38(+) and CD20(-). 39. A method of treating or alleviating a muscle disorder comprising administering to a subject a recombinant B cell according to any one of claims 35-38, wherein the muscle disorder is characterized by a decrease in follistatin. 40. A method for preventing a muscle disorder, comprising administering to a subject a recombinant B cell according to any one of claims 35-38, wherein the muscle disorder is characterized by a decrease in follistatin. 41. The method according to any one of paragraphs 13-29 or 39, 40, wherein the method comprises administering two or more sequential doses of genetically modified B cells to the subject. 42. The method of claim 41, wherein the administration comprises two or more doses of genetically modified B cells at suboptimal single dose concentrations. 43. The method of claim 41, wherein the administration comprises three or more doses of genetically modified B cells. 44. The method of claim 41, wherein the genetically modified B cells are autologous to the subject. 45. The method of claim 41, wherein the genetically modified B cells are allogeneic to the subject. 46. The method according to claim 41, wherein the subject is a human being. 47. The method according to claim 41, wherein the genetically modified B cells are CD20-, CD38- and CD138-. 48. The method according to claim 41, wherein the genetically modified B cells are CD20-, CD38+ and CD138+. 49. The method of claim 41, wherein the genetically modified B cells are CD20-, CD38+ and CD138-. 50. The method of claim 41, wherein the administration comprises intravenous, intraperitoneal, subcutaneous, intrathecal, intracameral, or intramuscular injection. 51. The method of claim 50, wherein the administration comprises intravenous injection. 52. The method according to any one of paragraphs 13-30 or 39-51, wherein the recombinant B cells are modified on day 2 or day 3 after culturing. 53. The method of claim 52, wherein the recombinant B cells are modified using a method comprising electroporation. 54. The method according to any of paragraphs 13-30 or 39-53, where (a) recombinant B cells are harvested for administration to a subject on a day ranging from day 1 to day 12 of in vitro culture. (b) the recombinant B cells are harvested for administration to the subject on day 4, day 5, day 6, or day 7, or day 8 in culture after modification. 55. The method according to any one of paragraphs 13-30 or 39-54, wherein the recombinant B cells are collected for administration to the subject on day 8 from the start of the culture or later, after modification. 56. The method of claim 55, wherein the recombinant B cells are collected for administration to the subject on day 10 from the start of the culture or earlier, after modification. 57. The method according to any one of claims 13-30 or 39-56, wherein the recombinant 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 culture period before and after modification. 58. The method of claim 57, wherein the multimerized CD40 ligand is a HIS-labeled CD40 ligand multimerized using an anti-his antibody. 59. The method of any one of paragraphs 13-30 or 39-58, further comprising expanding the recombinant B cells prior to administration to the subject. 60. The method of claim 59, wherein no more than 1% of the total final population of the expanded population of recombinant B cells produce the same B cell receptor sequence. 61. The method of claim 59, wherein any particular clone of B cells in the final population of expanded recombinant B cells constitutes less than 0.2% of the total B cell population. 62. The method of claim 59, wherein any particular clone of B cells in the final population of expanded recombinant B cells constitutes less than 0.05% of the total B cell population. 63. The method according to any one of claims 13-30 or 39-62, wherein the recombinant B cells comprise a polynucleotide encoding a selectable marker. 64. The method according to claim 63, wherein the selective marker is a human DHFR gene with increased resistance to methotrexate. 65. The method according to claim 64, wherein the human DHFR gene with increased resistance to methotrexate contains mutations of substitution of leucine to tyrosine at amino acid position 22 and phenylalanine to serine at amino acid position 31. 66. The method according to any one of claims 13-30 or 39-65, comprising treating the recombinant B cells with methotrexate prior to collection for administration. 67. The method according to claim 66, wherein methotrexate is administered in an amount of 100 nM to 300 nM. 68. The method according to claim 67, wherein methotrexate is administered in an amount of 200 nM. 69. The method of any one of paragraphs 13-30 or 39-68, wherein the recombinant B cells migrate to various tissues when administered to the subject. 70. The method of any one of claims 13-30 or 39-69, wherein at least one recombinant B cell from the population of recombinant B cells that is 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. 71. The method of claim 70, wherein at least one recombinant B cell from the population of recombinant B cells that is administered to the subject migrates to the bone marrow, intestine, muscle, spleen, kidney, heart, liver, lung, and brain of the subject. 72. The method of claim 28, 39 or 40, wherein the muscle disorder is selected from muscular dystrophy, inflammatory muscle disorder, muscle injury or trauma, muscle disuse and muscle atrophy or wasting. 73. The method of claim 72, wherein the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, or facioscapulohumeral muscular dystrophy. 74. The method of claim 72, wherein the inflammatory muscle disorder is inclusion body myositis. 75. The method according to claim 72, wherein muscle inactivity occurs after prolonged bed rest or immobilization of the limb. 76. The method of claim 72, wherein the muscle atrophy or weakening is caused by aging, malignancy, or chronic diseases. 77. The method of claim 28, 39 or 40, wherein the muscle disorder is sarcopenia. 78. The method of claim 28, 39 or 40, wherein the muscle disorder is spinal muscular atrophy (SMA). 79. The method of claim 28, 39 or 40, wherein the muscle disorder is amyotrophic lateral sclerosis (ALS). 80. The method of claim 28, 39 or 40, wherein the muscle disorder is Pompe disease. 81. The method according to any one of claims 72-80, wherein the follistatin comprises the amino acid sequence indicated in any one of SEQ ID NOs: 1-4.

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