HK40040375A - B cells genetically engineered to secrete follistatin and methods of using the same to treat follistatin-related diseases, conditions, disorders and to enhance muscle growth and strength - Google Patents

Description

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

Cross Reference to Related Applications

This application claims priority from U.S. provisional application No. 62/644,362 filed on day 3, 16, 2018 and U.S. provisional application No. 62/644,356 filed on day 3, 16, 2018, each of which is incorporated herein by reference in its entirety.

Statement regarding sequence listing

The sequence listing associated with the present application is provided in textual format in place of a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is IMCO-008-01 WO-ST25. txt. The text file was 12KB, created in 2019 on 3/18 and submitted electronically via EFS-Web.

Background

Technical Field

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

Description of the Related Art

Muscular Dystrophy (MD) is a progressive genetic neuromuscular disorder characterized by muscle atrophy and muscle weakness (Emery (2002) The Lancet,359: 687-. Many forms of muscular dystrophy are fatal and currently incurable.

Duchenne Muscular Dystrophy (DMD) is the most common X-linked neuromuscular disease. The disease is caused by a mutation in the DMD gene encoding dystrophin. This alteration or absence of protein results in abnormal myofascial tears. Abnormal changes in the diameter of muscle fibers (atrophic fibers and hypertrophic fibers) in the proximal muscle and sustained muscle damage are hallmarks of the disease. Damaged muscle releases the intracellular enzyme Creatine Kinase (CK). As a result, serum CK levels in DMD patients are characteristically high (up to 10-fold that of normal). The pathophysiological cascade is exacerbated by tissue inflammation, muscle fiber necrosis, and replacement of muscle with fibroadipose tissue.

Another allelic variant of the DMD gene causes a mild form of MD known as Becker Muscular Dystrophy (BMD). BMD is clinically similar to DMD, but the onset of symptoms occurs later in life.

Many agents have been tried in MD, but none have proven effective in preventing the course of the disease. Current treatment modalities are still in the areas of physical medicine and rehabilitation.

Many trials with corticosteroids (e.g., prednisone and/or its derivatives) have demonstrated improvement, particularly short term improvement, in individuals with MD. Although the exact mechanism by which corticosteroids alleviate the disease phenotype is not known, corticosteroids are thought to act by reducing inflammation, suppressing the immune system, improving calcium homeostasis, up-regulating the expression of compensatory proteins (compensatory proteins), and increasing myoblast proliferation (Khurana et al, (2003) nat. Rev. drug Discovery 2: 279-386). However, corticosteroids administered over time may induce muscle atrophy, which primarily affects the proximal muscle, i.e., the same muscle involved in DMD and BMD. Corticosteroid-induced muscle and other side effects may limit the long-term effectiveness of corticosteroid therapy.

The transforming growth factor-beta (TGF- β) superfamily comprises a variety of growth factors sharing common sequence elements and structural motifs. These proteins are known to exert biological effects on a variety of cell types in vertebrates and invertebrates. Members of the superfamily perform important functions in pattern formation and tissue specification during embryonic development 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 general branches: BMP/GDF and TGF- β/activin/BMP 10 branches, the members of which have different, often complementary, roles. By manipulating the activity of TGF- β family members, significant physiological changes in the organism can often be caused. For example, the piermonte cattle (Piedmontese) and belgium blue breeds carry loss-of-function mutations in the GDF8 (also known as myostatin) gene, which results in a significant increase in muscle mass. Grobet et al, nat. Genet.1997,17(1): 71-4. Furthermore, in humans, allelic inactivation of GDF8 is associated with increased muscle mass and reported abnormal strength. Schuelke et al, N Engl J Med 2004,350: 2682-8. In addition, mice genetically engineered to express dominant negative activin receptor IIB (ActRIIB) or to express follistatin have abnormal muscle mass (Lee, SJ and McPherron, AC, Proc Natl Acad Sci U S A.2001, 7/31; 98(16):9306-11), and overexpression of follistatin non-human primates enhances muscle growth and strength. Kota J et al, Sci Transl Med.2009, 11 months 11; 1(6).

Accordingly, there is a need for methods of delivering agents that function as potent modulators of TGF- β signaling.

Current methods for treating chronic diseases and conditions include direct infusion of therapeutic agents (e.g., therapeutic polypeptides), gene therapy via viral vectors, and adoptive transfer of stem cells (e.g., hematopoietic stem cell transfer). However, each of these methods has drawbacks. Infusion of recombinant therapeutic proteins suffers from the limited half-life of the protein, and all three methods provide sub-optimal tissue penetration of the therapeutic agent. Altering endogenous tissues to produce a therapeutic agent, such as via injection of recombinant adeno-associated virus (AAV) and lentiviral vectors, often results in production of the therapeutic agent from a centralized location. Generating the therapeutic agent from one location increases the likelihood of local toxicity in the tissue. In addition, since the recombinant virus is considered foreign, it is not possible to administer the viral vector multiple times without causing adverse effects, which means that there is only one injection opportunity to obtain the correct dose of the therapeutic agent. Given the biological variations inherent in the procedures (e.g., the use of viruses to introduce nucleic acids into cells in vivo), it would be very difficult to obtain the desired dose within the limits of a single injection.

Thus, there remains a need in the art for long-term treatment of many chronic diseases and disorders associated with TGF- β signaling.

Brief description of the embodiments

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

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 FST-344 splice site variant. In some embodiments, the B cell is a human B cell. In some embodiments, a B cell has been transduced with the follistatin gene. In some embodiments, the B cell comprises the follistatin gene in that the B cell has been transduced with the follistatin gene using the sleeping beauty transposon system. In some embodiments, the B cell expresses the follistatin gene as a result of being transduced with a virus that carries the follistatin gene. In some embodiments, the B cell comprises the follistatin gene in that the B cell has been transduced with a retrovirus, lentivirus, adenovirus, or adeno-associated virus comprising the follistatin gene. In some embodiments, the B cell is engineered to contain the follistatin gene using a targeted integration approach. In some embodiments, the targeted integration utilizes one or more of a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), and/or a CRISPR/Cas system including, but not limited to, the CRISPR/Cas9 system. In some embodiments, the B cell is engineered to comprise the follistatin gene by introducing a nucleic acid encoding follistatin 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 vector, non-viral DNA and/or RNA encoding follistatin introduced using chemical or physical means, such as lipofection, polycationic complexation, electroporation, etc. In some embodiments, the follistatin gene is secreted by the recombinant B cell. In some embodiments, the recombinant B cell is derived from a B cell obtained from a subject or a B cell derived from a cell obtained from a subject. In some embodiments, the recombinant B cell is derived from a B cell progenitor obtained from the subject. In some embodiments, the recombinant B cell is derived from a cell obtained from the subject that has dedifferentiated into the B cell or B cell progenitor. In some embodiments, the recombinant B cell is engineered by:

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

(b) transducing said cell with a DNA encoding said follistatin;

(c) expanding selected cells ex vivo; and

(d) (ii) differentiating said expanded cells ex vivo into plasma cells and/or plasmablasts.

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, electroporation utilizes the sleeping beauty turret subsystem. In some embodiments, the differentiated cells are CD38(+) and CD20 (-). In some embodiments, the present invention provides methods comprising administering such recombinant B cells to a subject.

In some embodiments, the present invention provides methods 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 provides a method of delivering follistatin to a subject in need thereof, comprising administering to the subject any one of the recombinant B cells expressing a follistatin polypeptide disclosed herein. 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, or facioscapulohumeral muscular dystrophy. In some embodiments, the muscle disorder is an inflammatory muscle disorder. In some embodiments, the inflammatory muscle disorder is inclusion body myositis. In some embodiments, the muscle disorder is a muscle injury or trauma. In some embodiments, the muscle disorder is muscle disuse. In some embodiments, the muscle disuse occurs after extended 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 disease. 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, administration of a B cell expressing a follistatin polypeptide to a subject with healthy muscle increases the muscle size or strength of the subject.

In some particular embodiments, the present disclosure provides methods for treating muscular dystrophy, comprising administering a B cell expressing a follistatin polypeptide to a subject having muscular dystrophy. In some embodiments, the subject has becker muscular dystrophy. In some embodiments, administering the recombinant B cells to the subject achieves treatment of a disease, disorder, or condition in the subject. In some embodiments, administering the recombinant B cell to the subject achieves treatment of muscular dystrophy. In some embodiments, administering the recombinant B cells to the subject causes the subject to gain weight. In some embodiments, the subject has increased body weight 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 the recombinant B cells to the subject causes the subject to acquire muscle. In some embodiments, administering the recombinant B cells to the subject causes the subject to become more robust. In some embodiments, the administration of the recombinant B cell results in an increase in plasma levels of follistatin in the subject.

In some embodiments, the present invention provides methods for treating, preventing, or ameliorating muscular dystrophy by administering a recombinant B cell comprising a follistatin gene. In some embodiments, a method of treating, preventing, or ameliorating muscular dystrophy comprises administering any of the recombinant B cells disclosed herein. In some embodiments, the method comprises administering two or more consecutive doses of genetically modified B cells to the subject. In some embodiments, administering comprises two or more doses of genetically modified B cells at sub-optimal single dose concentrations. In some embodiments, administering comprises three or more doses of genetically modified B cells. In some embodiments, the genetically modified B cell is 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 CD 138-. 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 CD 138-. In some embodiments, the administering comprises intravenous injection, intraperitoneal injection, subcutaneous injection, or intramuscular injection. In some embodiments, the administering comprises intravenous injection. In some embodiments, the genetically modified B cell is engineered at day 2 or day 3 after culture. In some embodiments, the genetically modified B cell is engineered using a method comprising electroporation. In some embodiments, the genetically modified B cells are harvested for administration to a subject on day 4, day 5, day 6, or day 7 of the post-engineering culture. In some embodiments, the genetically modified B cells are harvested for administration to a subject on or after day 8 of the post-engineering culture. In some embodiments, the genetically modified B cells are harvested for administration to a subject on day 10 or earlier of the post-engineering culture. In some embodiments, the harvested genetically modified B cells do not produce significant levels of inflammatory cytokines. In some embodiments, the genetically modified B cells are harvested at a culture time point determined that the genetically modified B cells do not produce significant levels of inflammatory 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 a multimerized CD40 ligand throughout the culture period before and after engineering. In some embodiments, the multimerized CD40 ligand is a HIS-labeled CD40 ligand that is multimerized using an anti-HIS antibody. In some embodiments, the method further comprises expanding the genetically modified B cells prior to administration to the subject. In some embodiments, the expanded final population of genetically modified B cells exhibits a high degree of polyclonality. In some embodiments, any particular B cell clone in the final population of expanded genetically modified B cells comprises less than 0.2% of the total B cell population. In some embodiments, any particular B cell clone in the final population of expanded genetically modified B cells comprises less than 0.05% of the total B cell population. In some embodiments, the genetically modified B cell comprises a polynucleotide encoding a human DHFR gene with enhanced resistance to methotrexate. In some embodiments, the human DHFR gene with increased resistance to methotrexate contains a leucine to tyrosine substitution mutation at amino acid 22 and a phenylalanine to serine substitution mutation at amino acid 31. In some embodiments, the method comprises treating the genetically modified B cells with methotrexate prior to harvesting for administration. In some embodiments, the methotrexate treatment is 100nM to 300 nM. In some embodiments, the methotrexate treatment is 200 nM. In some embodiments, the genetically modified B cells migrate to multiple tissues after administration to the subject. In some embodiments of the method, at least one genetically modified B cell of the population of genetically modified B cells administered to the subject migrates to one or more tissues selected from the group consisting of: bone marrow, intestine, muscle, spleen, kidney, heart, liver, lung, and brain. In some embodiments of the method, at least one genetically modified B cell in the population of genetically modified B cells administered to the subject migrates to bone marrow, intestine, muscle, spleen, kidney, heart, liver, lung, and brain of the subject.

In some embodiments, the present invention provides modified B cells transduced to express a follistatin gene and a dihydrofolate reductase (DHFR) gene.

Brief Description of Drawings

Figure 1 shows that treatment with B cells expressing follistatin results in increased follistatin levels in the plasma of mice. From left to right for each of the control and treatment groups: bars correspond to day 21, day 28 and day 35, respectively.

Figure 2 shows that plasma levels of follistatin in mice treated with B cells expressing follistatin correlate with levels of human IgG, which is a surrogate marker for transplantation. Figures 2A-2D show plasma levels of follistatin in four individual mice treated with B cells expressing follistatin.

Figure 3 shows the percent change in body weight of mice treated with or without B cells expressing follistatin.

Figure 4 shows the power assessment of mice treated with or without follistatin-expressing B-cells. Fig. 4A shows the force assessed by the foreleg grip test. Fig. 4B shows the force assessed by the four leg grip test. Fig. 4C shows the force evaluated by the suspension test. The percent improvement provided below the graph shows the average percent improvement for the treated group compared to the untreated group.

Figure 5 shows in vitro follistatin expression in follistatin-expressing B cells. Fig. 5A shows follistatin protein expression as determined by ELISA. Figure 5B shows follistatin mRNA expression determined by RT-PCR.

Detailed description of the invention

The practice of the present invention will employ, unless otherwise indicated explicitly to the contrary, conventional methods of molecular biology, recombinant DNA techniques, protein expression and protein/peptide/carbohydrate chemistry within the skill of the art, many of which are described below for purposes of illustration. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al, Molecular Cloning: A Laboratory Manual (3 rd edition, 2000); DNA Cloning: A Practical Approach, Vol.I & II (D.Glover edition); oligonucleotide Synthesis (n. gait editors, 1984); oligonucleotide Synthesis Methods and Applications (p. herewijn ed, 2004); nucleic Acid Hybridization (edited by B.Hames & S.Higgins, 1985); nucleic Acid Hybridization model Applications (edited by Buzdin and Lukyanov, 2009); transcription and transformation (edited by b.hames & s.higgins, 1984); animal Cell Culture (r. freshney, eds., 1986); freshney, R.I. (2005) Culture of Animal Cells, a Manual of Basic Technique, 5 th edition Hoboken NJ, John Wiley & Sons; b. perbal, a Practical Guide to Molecular Cloning (3 rd edition, 2010); farrell, R., RNA methods A Laboratory Guide for Isolation and Characterization (3 rd edition, 2005). The publications discussed above are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

Definitions and abbreviations

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used in the specification and the appended claims, the following terms take the meanings indicated, unless the contrary is indicated. In relation to this specification, a definition that is expressly defined herein is the correct definition for a term as defined herein, provided that the definition of that term differs from the definition given for the same term in the cited reference.

The terms "a" and "an" mean one or more unless specifically stated otherwise.

"about" means an amount, level, value, number, frequency, percentage, volume, size, amount, weight, or length that varies by as much as 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference amount, level, value, number, frequency, percentage, volume, size, amount, weight, 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.

A "composition" may comprise an active agent and an inert or active carrier, e.g., a pharmaceutically acceptable carrier, diluent or excipient. In particular embodiments, the compositions are sterile, substantially endotoxin-free or non-toxic to recipients at the dosages or concentrations employed.

Throughout this specification and claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be interpreted in an open and inclusive sense, i.e., "including but not limited to".

"consisting of … …" is intended to include and be limited to what is listed after the phrase "consisting. Thus, the phrase "consisting of … …" means that the listed elements are required or mandatory, and that no other elements may be present. "consisting essentially of … …" is intended to include any elements listed after the phrase and is limited to other elements that do not interfere with or facilitate the activity or action of the listed elements specified in this disclosure. Thus, the phrase "consisting essentially of … …" means that the listed elements are required or mandatory, but that other elements are optional, and that other elements may or may not be present depending on whether they affect the activity or action of the listed elements.

Throughout the specification, reference to "biological activity" or "biological activity" refers to any response, measured in vitro or induced in a cell, tissue, organ or organism (e.g., animal, or mammal, or human), as a result of administration of any of the compounds, agents, polypeptides, conjugates, pharmaceutical compositions contemplated herein. Biological activity may refer to agonism or antagonism. Biological activity may be a beneficial effect; or the biological activity may not be beneficial, i.e. toxic. In some embodiments, biological activity will refer to a positive or negative effect of a drug or pharmaceutical composition on a living subject (e.g., a mammal, such as a human). Thus, the term "biologically active" is intended to describe any compound having biological activity as described herein. Biological activity may be assessed by any suitable means currently known to the skilled person. Such assays may be qualitative or quantitative. The skilled person will readily appreciate that different assays need to be employed to assess the activity of different polypeptides; this is a daily task for the average researcher. Such assays are generally easy to perform in a laboratory setting with little requirement for optimization, and in general, commercial kits are available that provide simple, reliable, and reproducible readings of biological activity for a variety of polypeptides using a variety of techniques commonly used in most laboratories. When such kits are not available, the ordinarily skilled researcher can readily design and optimize an internal bioactivity assay for a target polypeptide without undue experimentation; as this is a routine aspect of the scientific process.

Reference to the term "such as" is intended to mean "such as but not limited to," and it should therefore be understood that what follows is merely an example of a particular embodiment and should in no way be construed as a limiting example. Unless otherwise indicated, the use of "as" is intended to expressly indicate that other embodiments have been contemplated and are encompassed by the present invention.

Reference throughout this specification to "an embodiment" or "one embodiment" or "an embodiment" or "some embodiments" or "certain 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 appearances of the phrases "in one embodiment" or "in an embodiment" or "in certain embodiments" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

An "increased" or "increased" amount is typically a "statistically significant" amount, and can include an increase of 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5,6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100 times, 500 times, 1000 times) (including all integers and decimal points between and greater than 1, such as 2.1, 2.2, 2.3, 2.4, etc.) that is an amount or level described herein. Similarly, an amount that is "reduced" or "less" is typically a "statistically significant" amount, and can include a reduction 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 times, 500 times, 1000 times) (including all integers and decimal points between and greater than 1, such as 1.5, 1.6, 1.7.1.8, etc.) that is an amount or level described herein.

The terms "in vitro", "ex vivo" and "in vivo" are intended herein to have their conventional scientific meaning. Thus, for example, "in vitro" means an experiment or reaction taking place with an isolated cellular component, such as an enzymatic reaction, for example, in a test tube using an appropriate substrate, enzyme, donor, and optionally buffer/cofactor. By "ex vivo" is meant an experiment or reaction performed using functional organs or cells that have been removed from an organism or propagated independently of the organism. By "in vivo" is meant an experiment or reaction that occurs within a normal, intact living organism.

"mammal" includes humans and includes domestic animals such as laboratory animals and domestic pets (e.g., cats, dogs, pigs, cattle, sheep, goats, horses, and rabbits) as well as non-domestic animals such as wild animals and the like.

"optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

"pharmaceutical composition" refers to a formulation of a compound (e.g., a therapeutically useful polypeptide) with a vehicle generally accepted in the art for delivering the compound to an animal (e.g., a human). Such a medium may therefore comprise any pharmaceutically acceptable carrier, diluent or excipient.

"pharmaceutically effective excipients" and "pharmaceutically effective carriers" are well known to those skilled in the art and methods for their preparation will be apparent to those skilled in the art. Such compositions and methods for their preparation can be found in Remington's Pharmaceutical Sciences, 19 th edition (Mack Publishing Company,1995, incorporated herein).

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

As used herein, a "subject" includes any animal exhibiting or at risk of exhibiting a disease or condition that can be treated with an agent of the invention. Suitable subjects include laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals, and domestic or pet animals (e.g., cats or dogs). Including non-human primates, and preferably human patients.

"substantially" or "substantially" means a sufficient or substantial amount, quantity, size; almost completely or completely; for example, 95% or higher of some given number.

"therapeutic agent" 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 effecting the treatment of a disease or condition as defined below.

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

As used herein, "treatment" or "treatment" encompasses treatment of a target disease or condition in a subject, preferably a human, having the target disease or condition, and includes: (i) preventing or inhibiting the occurrence of the disease or condition in a subject, particularly when such a subject is susceptible to, but has not yet been diagnosed as having, the condition; (ii) inhibiting the disease or condition, i.e., arresting its development; (iii) alleviating, i.e., causing regression of, the disease or condition; or (iv) alleviating a symptom caused by the disease or condition. As used herein, the terms "disease," "disorder," and "condition" may be used interchangeably, or may be different, in that a particular disease, injury, or condition may not have a known causative agent (such that the cause has not yet been identified), and thus has not yet been considered an injury or disease, but merely an undesirable condition or syndrome, with more or less of a particular set of symptoms having been identified by a clinician.

SUMMARY

The invention relates inter alia to the production of follistatin from autologous and/or allogeneic B cells that have been altered by the introduction of nucleic acids and also to methods of administering 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 that comprise one or more nucleic acids (e.g., a transgene) to produce follistatin (e.g., a transgene that enables expression of a follistatin polypeptide, such as a therapeutic follistatin polypeptide). In particular, the modified B cells can be administered in a single dose or multiple doses.

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

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

B cell

Upon leaving the bone marrow, B cells act as Antigen Presenting Cells (APCs) and internalize antigen. The antigen is taken up by B cells via receptor-mediated endocytosis and processed. The antigen is processed into antigenic peptides, loaded onto MHC II molecules, and presented to CD4+ T helper cells on the extracellular surface of B cells. These T cells bind MHC II/antigen molecules and cause B cell activation. Upon stimulation by T cells, activated B cells begin to differentiate into more specialized cells. Germinal center B cells can differentiate into long-lived memory B cells or plasma cells. In addition, secondary immune stimulation can cause memory B cells to produce additional plasma cells. Plasma formation from memory or non-memory B cells is preceded by the formation of precursor plasmablasts that eventually differentiate into plasma cells that produce large amounts of antibodies (see Trends Immunol.2009, 6 months; 30(6): 277-285; Nature Reviews,2005,5: 231-242). Plasmablasts secrete more antibodies than B cells, but less than plasma cells. They divide rapidly, continue to internalize and present antigen to T cells. Plasmablasts have the ability to migrate to sites of chemokine production (as in bone marrow) so that they can differentiate into long-lived plasma cells. Finally, the plasmablast can remain as a plasmablast for several days and then die or irreversibly differentiates into mature, fully differentiated plasma cells. In particular, plasmablasts that can home to tissues containing a plasma cell niche (e.g., in the bone marrow) can replace resident plasma cells, thereby becoming long-lived plasma cells that can continue to secrete high levels of proteins for many years.

B cells (e.g., to express follistatin) used in the methods described herein include pan B cells, memory B cells, plasmablasts, and/or plasma cells. In one embodiment, the modified B cell is a memory B cell (e.g., modified to express follistatin). In one embodiment, the modified B cell is a plasmablast (e.g., modified to express follistatin). In one embodiment, the modified B cell is a plasma cell (e.g., which is modified to express follistatin).

Terminally differentiated plasma cells typically do not express common pan B cell markers such as CD19 and CD20, and express relatively few surface antigens. Plasma cells express CD38, CD78, CD138, and interleukin-6 receptor (IL-6R), and lack CD45 expression, and these markers can be used to identify plasma cells, e.g., by flow cytometry. CD27 is also a good marker for plasma cells because the primary B cells are CD27-, the memory B cells are CD27+ and the plasma cells are CD27+ +. Memory B cell subsets can also express surface IgG, IgM, and IgD, whereas 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, The "plasmid cell" Page Version ID: 404969441; last date of revision: retrieval of 12.30.2010, 54UTC, 4.2011; see also Jourdan et al, blood.2009, 12.10.114 (25), 5173-81; Trends Immunol.2009, 6.2009; 30(6), 277-285; Nature Reviews,2005,5: 231-242; Nature Med.2010,16: 123-129; Neuberger, M.S., Honjoo, T.; Alt, Frederick W. (Molecular of B cell Am., catalog, M.S., H.S.; J.S.; mineral; Jarke J.S. J.; mineral, moisture, Ga. G, moisture, pp. 12, page 387-; rawstron AC (5.2006.) "Immunophenotyping of plasma cells". Curr Protoc Cytom).

As used herein, "quiescent" refers to a state of a cell in which the cell is not actively proliferating.

As used herein, "activated" refers to a cellular state in which cells actively proliferate and/or produce cytokines in response to a stimulus.

As used herein, the terms "differentiation" and "differentiated" refer to a change in a cell phenotype from one cell type or state to another. For example, memory B cells transformed into plasma cells are differentiated.

The term "subject" is intended to include living organisms (e.g., mammals) that can elicit an adaptive immune response. 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 (PBMCs), bone marrow, lymph node tissue, cord blood, tissue at the site of infection, spleen tissue, and tumors. In a preferred embodiment, the source of B cells is PBMCs. In certain embodiments of the present disclosure, a number of B cell lines available in the art may be used.

In certain embodiments of the methods described herein, B cells can use a number of techniques known to those of skill in the art, such as FICOLL^TM(copolymers of sucrose and epichlorohydrin that can be used to prepare high density solutions) are obtained from blood units collected from a subject. In a preferred embodiment, the cells from the circulating blood of the individual are obtained by apheresis or leukapheresis. The products of apheresis typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells collected by apheresis may be washed to remove plasma fractions and placed in an appropriate buffer or culture medium for subsequent processing steps. In one embodiment of the methods described herein, the cells are washed with Phosphate Buffered Saline (PBS). In alternative embodiments, the wash solution lacks calcium and may lack magnesium or may lack many, if not all, divalent cations. As one of ordinary skill in the art will readily appreciate, the washing step can be accomplished by methods known to those of skill in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor) according to the manufacturer's instructions. After washing, the cells can be resuspended in various biocompatible buffers such as, for example, PBS. Alternatively, the sample from the apheresis procedure may be freed of undesired components and the cells resuspended directly in the culture medium.

B cells can be isolated from peripheral blood or leukapheresis using techniques known in the art. For example, FICOLL may be used^TM(Sigma-Aldrich, St Louis, Mo.) isolation of PBMC and use of any of a number of antibodies known in the art, such as the Rosette tetramer complex system (stemCell Technologies, Vancouver, Canada) or MACS^TMMicroBead Technology (Miltenyi Biotec, San Diego, Calif.) CD19+ B cells were purified by negative or positive selection. In certain embodiments, memory B cells are isolated as described by Jourdan et al (blood.2009, 12/10; 114(25): 5173-81). For example, after removal of CD2+ cells using anti-CD 2 magnetic beads, the cells can be washedCD19+ CD27+ memory B cells were sorted by FACS. Bone Marrow Plasma Cells (BMPCs) may be purified using anti-CD 138 magnetic microbead sorting or other similar methods and reagents. Human B cells can be isolated, for example, using CD19 MicroBeads, human (Miltenyi Biotec, San Diego, CA). Human Memory B cells can be isolated, for example, by human (Memory B Cell Isolation Kit, human) (Miltenyi Biotec, San Diego, CA) using a Memory B Cell Isolation Kit.

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

In one embodiment, gradient-based purification (e.g., FICOLL) is used^TM) PBMCs were obtained from blood samples. In another embodiment, the PBMCs are obtained from a apheresis-based collection. In one embodiment, the B cells are isolated from PBMCs by isolating pan-B cells. The isolation step may utilize positive and/or negative selection. In one embodiment, negative selection comprises depleting T cells using anti-CD 3 conjugated microbeads, thereby providing a T cell depleted fraction. In a further embodiment, memory B cells are isolated from pan B cell or T cell depleted fractions by positive selection for CD 27.

In a particular embodiment, memory B cells are isolated by depleting unwanted cells and then positively selecting with CD27 MicroBeads. A mixture of biotinylated antibodies and anti-biotin microbeads directed to CD2, CD14, CD16, CD36, CD43, and CD235a (glycophorin a) can be used to deplete unwanted cells such as T cells, NK cells, monocytes, dendritic cells, granulocytes, platelets, and erythroid cells.

In one embodiment, transformed memory B cells are obtained. As used herein, "transformed memory B cells" or "transformed B cells" refer to B cells that have undergone isotype class switching. In one embodiment, the transformed memory B cells are positively selected for IgG. In another embodiment, the transformed memory B cells are obtained by depleting cells expressing IgD and IgM. Transformed Memory B cells can be isolated, for example, by human (Switched Memory B Cell Kit, human) (Miltenyi Biotec, San Diego, CA) using a transformed Memory B Cell Kit.

For example, in one particular embodiment, non-target cells may be labeled with a mixture of biotinylated CD2, CD14, CD16, CD36, CD43, CD235a (glycophorin a), anti-IgM, and anti-IgD antibodies. These cells can then be magnetically labeled with anti-biotin microbeads. Transformed memory B cells of high purity can be obtained by depleting magnetically labeled cells.

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

In vitro culture of B cells

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 will be appreciated by those skilled in the art, plasma cells can be identified by cell surface protein expression patterns using standard flow cytometry methods. For example, terminally differentiated plasma cells express relatively few surface antigens and do not express common pan B cell markers such as CD19 and CD 20. In contrast, plasma cells can be identified by expression of CD38, CD78, CD138 and IL-6R and lack of expression of CD 45. CD27 can also be used to identify plasma cells because the naive B cells are CD27 ", the memory B cells are CD27+, and the plasma cells are CD27+ +. Plasma cells express high levels of CD38 and CD 138.

In one embodiment, the B cell is a CD 138-memory B cell. In one embodiment, the B cell is a CD138+ plasma cell. In one embodiment, the B cells are activated and have a cell surface phenotype of CD138-, CD27 +.

In one embodiment, the B cell is a CD20, CD 138-memory B cell. In one embodiment, the B cell is a CD20 ", CD138+ plasma cell. In one embodiment, the B cells are activated and have a cell surface phenotype of CD20-, CD138-, CD27 +.

In one embodiment, the B cell is a CD20-, CD38-, CD 138-memory B cell. In one embodiment, the B cell is a CD20-, CD38+, CD138+ plasma cell. In one embodiment, the B cells are activated and have a cell surface phenotype of CD20-CD38-CD138-CD27 +.

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 199892: 4509-4520; Luo, et al, Blood 2009113: 1422-1431). Such factors may be selected from, but are not limited to, the following: 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-, C-type chemokines XCL1 and XCL2, C-type chemokines (including CCL1-CCL28 to date) and CXC chemokines (including CXCL1-CXCL17 to date) TNF superfamily members (e.g., TNF- α, 4-1BB ligand, B cell activating factor (BLyS), FAS ligand, sCD40L (including multimeric forms of sCD 40L; e.g., histidine-tagged soluble recombinant CD40L in combination with anti-polyhistidine mAb to cluster multiple sCD40L molecules together), lymphotoxins, OX40L, RANKL, TRAIL), CpG, and other toll-like receptor agonists (e.g., CpG).

B cell activating factors can be added to in vitro cell cultures at various concentrations to achieve desired results (e.g., expansion or differentiation). In one embodiment, the B cell activating factor is used to expand cultured B cells. In one embodiment, the B cell activating factor is used to differentiate cultured B cells. In another embodiment, the B cell activating factor is used to expand and differentiate cultured B cells. 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 may be 1) used to expand B cells but not B cells, 2) used to differentiate B cells but not B cells, or 3) used to expand and differentiate B cells.

For example, in some embodiments, B cells are cultured in B cell culture media containing one or more B cell activating factors selected from the group consisting of CD40L, IL-2, IL-4, and IL-10 to expand the B cells. In one embodiment, B cells are cultured at 0.25-5.0 μ g/ml CD 40L. In one embodiment, the concentration of CD40L is 0.5. mu.g/ml. In one embodiment, a cross-linking agent (such as an anti-HIS antibody in combination with HIS-labeled CD 40L) is used to generate multimers of CD 40L. In one embodiment, molecules of CD40L are covalently linked or held together using a protein multimerization domain (e.g., the Fc region or leucine zipper domain of IgG). In one embodiment, CD40L is conjugated to a bead. In one embodiment, CD40L is expressed by feeder cells. In one embodiment, B cells are cultured in the presence of 1-10ng/ml IL-2. In one embodiment, the concentration of IL-2 is 5 ng/ml. In one embodiment, B cells are cultured in the presence of 1-10ng/ml IL-4. In one embodiment, the concentration of IL-4 is 2 ng/ml. In one embodiment, B cells are cultured in the presence of 10-100ng/ml IL-10. In one embodiment, the concentration of IL-10 is 40 ng/ml.

In one embodiment, B cells are expanded by culturing the B cells in a B cell culture medium comprising one or more B cell activating factors selected from the group consisting of CD40L, IL-2, IL-4, IL-10, IL-15, and IL-21. In one embodiment, B cells are cultured at 0.25-5.0 μ g/ml CD 40L. In one embodiment, the concentration of CD40L is 0.5. mu.g/ml. In one embodiment, a cross-linking agent (such as an anti-HIS antibody in combination with HIS-labeled CD 40L) is used to generate multimers of CD 40L. In one embodiment, molecules of CD40L are covalently linked or held together using a protein multimerization domain (e.g., the Fc region or leucine zipper domain of IgG). In one embodiment, CD40L is conjugated to a bead. In one embodiment, CD40L is expressed by feeder cells. In one embodiment, B cells are cultured in the presence of 1-10ng/ml IL-2. In one embodiment, the concentration of IL-2 is 5 ng/ml. In one embodiment, B cells are cultured in the presence of 1-10ng/ml IL-4. In one embodiment, the concentration of IL-4 is 2 ng/ml. In one embodiment, B cells are cultured in the presence of 10-100ng/ml IL-10. In one embodiment, the concentration of IL-10 is 40 ng/ml. In one embodiment, B cells are cultured in the presence of 50-150ng/ml IL-15. In one embodiment, the concentration of IL-15 is 100 ng/ml. In one embodiment, B cells are cultured in the presence of 50-150ng/ml IL-21. In one embodiment, the concentration of IL-21 is 100 ng/ml. In a particular embodiment, B cells are cultured in a B cell culture medium comprising CD40L, IL-2, IL-4, IL-10, IL-15, and IL-21 for expansion of the B cells.

For example, in one embodiment, B cells are cultured with B cell culture media containing the B cell activating factors CD40L, IL-2, IL-4, IL-10, IL-15, and IL-21 for expansion of the B cells, wherein CD40L is crosslinked with a crosslinking agent to produce multimers of CD 40L. Such culture systems can be maintained throughout a culture period (e.g., a 7 day culture period) in which B cells are transfected or otherwise engineered to express a transgene of interest (e.g., an exogenous polypeptide such as, for example, FST). The transgene may be integrated into the B cell (e.g., via a viral or non-viral vector). The transgene can be expressed in B cells via the use of transposons. The transgene can be expressed in B cells due to targeted integration of the transgene into the genome of the B cells. Targeted integration may be via homologous recombination. Homologous recombination can occur at double-strand breaks induced by nucleases. The nuclease can be, e.g., a zinc finger nuclease, a TALE nuclease (TALEN), a meganuclease (e.g., a homing endonuclease), or via a CRISPR/CAS9 nuclease system.

In another example, in one embodiment, B cells are cultured in a B cell culture medium containing one or more B cell activating factors selected from the group consisting of CD40L, IFN- α, IL-2, IL-6, IL-10, IL-15, IL-21, and P-class CpG oligodeoxynucleotides (P-ODN) to differentiate the B cells. In one embodiment, B cells are cultured at 25-75ng/ml CD 40L. In one embodiment, the concentration of CD40L is 50 ng/ml. In one embodiment, the B cells are cultured in the presence of 250-750U/ml IFN-. alpha.s. In one embodiment, the concentration of IFN- α is 500U/ml. In one embodiment, the B cells are cultured in the presence of 5-50U/ml IL-2. In one embodiment, the concentration of IL-2 is 20U/ml. In one embodiment, B cells are cultured in the presence of 25-75ng/ml IL-6. In one embodiment, the concentration of IL-6 is 50 ng/ml. In one embodiment, B cells are cultured in the presence of 10-100ng/ml IL-10. In one embodiment, the concentration of IL-10 is 50 ng/ml. In one embodiment, B cells are cultured in the presence of 1-20ng/ml IL-15. In one embodiment, the concentration of IL-15 is 10 ng/ml. In one embodiment, B cells are cultured in the presence of 10-100ng/ml IL-21. In one embodiment, the concentration of IL-21 is 50 ng/ml. In one embodiment, B cells are cultured in the presence of 1-50. mu.g/ml p-ODN. In one embodiment, the concentration of p-ODN is 10. mu.g/ml.

In one embodiment, the B cells are contacted with or cultured on feeder cells. In one embodiment, the feeder cells are stromal cell lines, such as murine stromal cell line S17 or MS 5. In another embodiment, the isolated CD19+ cells are cultured in the presence of fibroblasts expressing CD40 ligand (CD40L, CD154) in the presence of one or more B cell activator cytokines such as IL-10 and IL-4. In one embodiment, CD40L is provided bound to a surface, such as a tissue culture plate or bead. In another embodiment, purified B cells are cultured in the presence or absence of feeder cells, in the presence of CD40L and one or more cytokines or factors selected from the group consisting of IL-10, IL-4, IL-7, p-ODN, CpG DNA, IL-2, IL-15, IL6, and IFN- α.

In another embodiment, the B cell activating factor is provided by transfection into B cells or other feeder cells. In this context, one or more factors that promote the differentiation of B cells into antibody-secreting cells and/or one or more factors that increase the lifespan of antibody-producing cells may be used. Such factors include, for example, Blimp-1, TRF4, anti-apoptotic factors such as Bcl-xl or Bcl5, or constitutively active mutants of the CD40 receptor. In addition, factors that promote the expression of downstream signaling molecules, such as TNF receptor-related factor (TRAF), may also be used in the activation/differentiation of B cells. In this regard, the cell activation, cell survival and anti-apoptotic functions of the TNF receptor superfamily are primarily mediated by TRAF1-6 (see, e.g., r.h. arch, et al, Genes dev.12(1998), pages 2821-2830). Downstream effectors of TRAF signaling include transcription factors in the NF-. kappa.B and AP-1 families, which open genes involved in various aspects of cellular and immune function. Furthermore, it has been shown that activation of NF-. kappa.B and AP-1 can provide cellular protection from apoptosis via transcription of anti-apoptotic genes.

In another embodiment, an epstein-barr virus (EBV) -derived protein is used to activate and/or differentiate B cells or to increase the lifespan of antibody producing cells. 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.

In some casesIn embodiments, contacting a B cell with a B cell activator using the methods provided herein results in, inter alia, cell proliferation (i.e., expansion), modulation of lgM + cell surface phenotype to a phenotype consistent with activated mature B cells, Ig secretion, and isotype switching. Known and commercially available cell isolation kits such as MiniMACS can be used^TMCell isolation System (Miltenyi Biotech, Bergisch Gladbach, Germany) isolated CD19+ B cells. In certain 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 certain embodiments, the method comprises culturing B cells with transformed stromal cells (e.g., MS5) in the presence of one or more of the above-described factors, thereby providing low levels of anchored CD40L and/or CD40L bound to the plates or beads.

As discussed above, B cell activating factors induce the expansion, proliferation or differentiation of B cells. Thus, B cells are contacted with one or more of the B cell activating factors listed above to obtain an expanded cell population. The cell population may be expanded prior to transfection. Alternatively or additionally, the cell population may be expanded following 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, 20127 (12): e 51946). In one embodiment, expanding the B cell population comprises culturing the cells with IL-2, IL-10, CpG, and CD 40L. In one embodiment, expanding the B cell population comprises culturing the cells with IL-2, IL-4, IL-10, IL-15, IL-21, and CD 40L. 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 CD 40L.

In another embodiment, expansion of the B cell population is induced and/or enhanced by a transgene introduced into the B cells. For example, B cells containing a recombinant receptor or an engineered receptor induce a cellular signaling pathway (e.g., downstream of signaling by CD40) upon binding of their ligand (e.g., a soluble ligand or a cell surface-expressed ligand). In one embodiment, the B cells overexpress CD40 due to expression of the CD40 transgene. In another embodiment, the B cell expresses an engineered receptor, including, e.g., 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, such as CD 40.

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

As known to those skilled in the art, any of a variety of media may be used in the methods of the present invention (see, e.g., Current Protocols in Cell Culture,2000-2009, John Wiley & Sons, Inc.). In one embodiment, the medium used in the methods described herein includes, but is not limited to, an Iskoff's modified Dulbecco's medium (with or without fetal bovine or other suitable serum). Illustrative media also include, but are not limited to, IMDM, RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo15, and X-Vivo 20. In further embodiments, the culture medium may comprise surfactants, antibodies, human plasma protein powder or reducing agents (e.g., N-acetyl-cysteine, 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 crosslinking enhancer may also be used.

B cells are cultured under conditions and for a sufficient period of time to achieve the desired differentiation and/or activation. In certain embodiments, B cells are cultured and cultured under conditions and for a sufficient period of time such that 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% of the B cells differentiate and/or activate as desired. In one embodiment, B cells are activated and differentiate into a mixed population of plasmablasts and plasma cells. As will be appreciated by those skilled in the art, standard flow cytometry methods as described elsewhere herein may be usedBy cell surface protein expression patterns, e.g., CD38, CD78, IL-6R, CD27^{Height of}And expression of one or more of CD138 and/or lack or reduction of expression of one or more of CD19, CD20, and CD45 to identify plasmablasts and plasma cells. As one skilled in the art will recognize, memory B cells are typically CD20+ CD19+ CD27+ CD38 ", while early plasmablasts are CD20-CD19+ CD27+ + CD38+ +. In one embodiment, the cells cultured using the methods described herein are CD20-, CD38+, CD 138-. In another embodiment, the cell has the phenotype CD20-, CD38+, CD138 +. In certain embodiments, the cells are cultured for 1-7 days. In further embodiments, the cells are cultured for 7 days, 14 days, 21 days, or more. Thus, the cells can be cultured under appropriate conditions for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days or more. The cells are replated and media and supplements may be added or replaced as needed using techniques known in the art.

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

By such as³H-uridine incorporation into RNA (RNA synthesis increases with B cell differentiation) or by³The technique of H-thymidine incorporation (which measures DNA synthesis associated with cell proliferation) measures the induction of B-cell activation. In one embodiment, interleukin-4 (IL-4) may be added to the culture medium at an appropriate concentration (e.g., about 10ng/ml) to enhance B cell proliferation.

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., 10ng/ml) and IL-5 (e.g., 5ng/ml) or other B cell activating cytokines. Flow cytometry can also be used to measure cell surface markers characteristic 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 Monoyeroid Lineages in Normal Human Bone Marrow, in Flow Cytometry in Hematology, Laerum OD, Bjerksnes R. eds, Academic Press, New York 1992; page 31-page 42; and LeBein TW et al, leukamia 1990; 4:354-358.

After culturing for a suitable period of time, such as, for example, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or more (typically about 3 days), an additional volume of culture medium may be added. Supernatants from each culture were harvested at various time points during culture and quantified for IgM and IgG1 as described in Noelle et al (1991) J.Immunol.146: 1118-. In one embodiment, the culture is harvested and measured for expression of the transgene of interest using flow cytometry, enzyme linked immunosorbent assay (ELISA), ELISPOT, or other assays known in the art.

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

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

Engineering of B cells

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

In one embodiment, genetically modified B cells are transfected with a transgene. In a particular embodiment, the genetically modified B cell has a follistatin transgene.

Exemplary methods for transfecting B cells are provided in WO 2014/152832 and WO 2016/100932, both of which are incorporated herein by reference in their entirety. Transfection of B cells can be accomplished using any of a variety of methods available in the art for introducing DNA or RNA into B cells. Suitable techniques may include calcium phosphate transfection, DEAE-dextran, electroporation, pressure-mediated transfection or "cell extrusion" (e.g., CellSqueeze microfluidic systems, SQZ Biotechnologies), nanoparticle-mediated or liposome-mediated transfection and transduction using retroviruses or other viruses such as vaccinia. See, e.g., Graham et al, 1973, Virology 52: 456; sambrook et al, 2001, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratories; davis et al, 1986, Basic Methods in Molecular Biology, Elsevier; chu et al, 1981, Gene 13: 197; US 5,124,259; US 5,297,983; US 5,283,185; US 5,661,018; US6,878,548; US 7,799,555; US 8,551,780; and US 8,633,029. An example of a commercially available electroporation technique suitable for B cells is Nucleofector^TMTransfection techniques.

Transfection may be performed before or during in vitro culture of the isolated B cells in the presence of one or more of the activation and/or differentiation factors described above. For example, cells are transfected on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, day 21, day 22, day 23, day 24, day 25, day 26, day 27, day 28, day 29, day 30, day 31, day 32, day 33, day 34, day 35, day 36, day 37, day 38, or day 39 of in vitro culture. In one embodiment, the cells are transfected on day 1, day 2, or day 3 of in vitro culture. In a particular embodiment, cells are transfected on day 2. For example, cells are electroporated on day 2 of in vitro culture for delivery of, e.g., plasmids, transposons, micro-loops, or self-replicating RNA. In another embodiment, the cells are transfected at day 4, day 5, day 6, or day 7 of in vitro culture. In particular embodiments, the cells are transfected at day 6 of in vitro culture. In another embodiment, the cells are transfected on day 5 of in vitro culture.

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

In one embodiment, the non-viral vector is used to deliver DNA or RNA (e.g., DNA or RNA comprising a sequence encoding a follistatin polypeptide) to memory B cells and/or plasma cells. For example, systems that do not require viral integration systems to facilitate transfection of memory B cells and/or plasma cells include, but are not limited to, transposons (e.g., sleeping beauty or other transposable systems such as Piggybac), Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), meganucleases, minicircles, replicons, artificial chromosomes (e.g., bacterial artificial chromosomes, mammalian artificial chromosomes, and yeast artificial chromosomes), plasmids, cosmids, and phages.

In some embodiments, such virus-independent vector systems can also be delivered via viral vectors known in the art or described below. For example, in some embodiments, viral vectors (e.g., retroviruses, lentiviruses, adenoviruses, adeno-associated viruses) are used to deliver one or more non-viral vectors (e.g., one or more of the Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs), meganucleases, or any other enzyme/complementary vector, polynucleotide, and/or polypeptide capable of promoting targeted integration, as described above TALEN, CRISPR/Cas, meganuclease) cleaves the endogenous locus and a follistatin transgene is administered to the cell such that the transgene is integrated into the endogenous locus and expressed in the cell. The follistatin transgene may be included in a donor sequence that is integrated into the DNA of the host cell at or near the site of nuclease cleavage.

Integration of a foreign sequence (e.g., a sequence encoding a follistatin polypeptide) can occur via recombination. As will be clear to one skilled in the art, "recombination" refers to the process of genetic information exchange between two polynucleotides, including but not limited to donor capture by non-homologous end joining (NHEJ) and homologous recombination. The recombination may be homologous recombination. For the purposes of the present disclosure, "Homologous Recombination (HR)" refers to a special form of such exchange, for example, in the modification via homology-directed repair mechanismsDouble strand breaks in the double cells occur. This process exploits the homology of nucleotide sequences, whereby a "donor" molecule (e.g., a donor polynucleotide sequence or donor vector comprising such a sequence) is used by the DNA repair machinery of the cell as a template to repair a "target" molecule (i.e., a molecule that undergoes a double-strand break), and by these means causes the transfer of genetic information from the donor to the target. In some embodiments of HR-directed integration, the donor molecule may contain at least 2 regions of homology to the genome ("homology arms"). In some embodiments, the homology arms can be, for example, at least 50-100 base pairs in length. The homology arms can have significant DNA homology to the region of genomic DNA flanking the cleavage site where targeted integration is to occur. The homology arms of the donor molecule can flank the DNA (e.g., comprising a DNA encoding follistatin) to be integrated into the target genome or target DNA locus. Chromosome breakage, followed by repair using the homologous regions of the plasmid DNA as templates, may result in the transfer of the inserted transgene flanked by homologous arms into the genome. See, e.g., Koller et al (1989) Proc. nat' l. Acad. Sci. USA 86(22): 8927-; thomas et al (1986) Cell 44(3): 419-428. The frequency of this type of homotactic targeted integration can be increased by up to 10 by deliberate generation of double-strand breaks in the vicinity of the target region⁵Kyagar (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.

In accordance with the present disclosure, 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., by recombination such as HR) can be used to engineer a cell (e.g., a memory B cell or a plasmablast).

Double-stranded breaks (DSBs) or nicks can be created by site-specific nucleases such as Zinc Finger Nucleases (ZFNs), TAL effector domain nucleases (TALENs), meganucleases, or by directing specific cleavage with engineered crRNA/trace RNA (single guide RNA) using the CRISPR/Cas9 system. See, e.g., Burgess (2013) Nature Reviews Genetics 14:80-81, Urnov et al (2010) Nature 435(7042) 646-51; U.S. patent publication 20030232410; 20050208489, respectively; 20050026157, respectively; 20050064474; 20060188987; 20090263900, respectively; 20090117617, respectively; 20100047805, respectively; 20110207221, respectively; 20110301073 and international publication WO 2007/014275, the disclosures of which are incorporated by reference in their entirety for all purposes.

In some embodiments, a cell (e.g., a memory B cell or a plasmablast) is engineered via zinc finger nuclease-mediated targeted integration of a donor construct (e.g., a follistatin donor construct). Zinc Finger Nucleases (ZFNs) are enzymes that are capable of specifically recognizing and cleaving a target nucleotide sequence due to the coupling of a "zinc finger DNA binding protein" (ZFP) (or binding domain) to the nuclease by one or more zinc fingers binding DNA in a sequence-specific manner. The ZFN may comprise any suitable cleavage domain (e.g., nuclease) operably linked to the ZFP DNA binding domain to form an engineered ZFN that promotes site-specific cleavage of the target DNA sequence (see, e.g., Kim et al (1996) Proc Natl Acad Sci USA93(3): 1156-1160). For example, a ZFN may comprise a target-specific ZFP linked to a FOK1 enzyme or a portion of a FOK1 enzyme. In some embodiments, a ZFN used in a ZFN-mediated targeted integration method utilizes two separate molecules, each molecule comprising a subunit of FOK1 enzyme that each binds a ZFP, each ZFP specific for a DNA sequence flanking the target cleavage site, and when two ZFPs bind their respective target DNA sites, the FOK1 enzyme subunits are brought into proximity and bind together, thereby activating nuclease activity that cleaves the target cleavage site. ZFNs have been used for genome modification in a variety of organisms (e.g., U.S. patent publication 20030232410; 20050208489; 20050026157; 20050064474; 20060188987; 20060063231; and international publication WO 07/014,275, which are incorporated herein by reference in their entirety). Custom ZFPs and ZFNs are commercially available from, e.g., Sigma Aldrich (st. louis, MO), and any location of DNA can be routinely targeted and cleaved using such custom ZFNs.

In some embodiments, the cell (e.g., memory B cell or plasmablast) is engineered via CRISPR/Cas (e.g., CRISPR Cas9) nuclease-mediated integration of a donor construct (e.g., follistatin donor construct). CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR associated) nuclease systems are engineered nuclease systems that can be used for genome engineering based on bacterial systems. It is based on part of the adaptive immune response of many bacteria and archaea. When a virus or plasmid invades a bacterium, the DNA segment of the invader is converted to CRISPR RNA (crRNA) by an "immune" response. This crRNA then associates with another type of RNA called tracrRNA through a partially complementary region to direct the Cas9 nuclease to a region of homology to the crRNA in the target DNA (called the "pro-spacer"). Cas9 cleaves the DNA to create a blunt end at the DSB at the site specified by the 20 nucleotide guide sequence contained within the crRNA transcript. Cas9 requires both crRNA and tracrRNA for site-specific DNA recognition and cleavage. The system has now been engineered such that the crRNA and tracrRNA can be combined into one molecule ("single guide RNA"), and the crRNA equivalent of the single guide RNA can be engineered to guide Cas9 nuclease to any desired sequence (see Jinek et al (2012) Science 337, p. 816-821 Jinek et al, (2013), ebife 2: e00471, and David Segal, (2013) ebife 2: e 00563). Thus, the CRISPR/Cas system can be engineered to produce DSBs at the desired target of the genome, and repair of DSBs can be affected by causing increased error-prone repair using repair inhibitors. As will be clear to the skilled person, other CRISPR nucleases besides Cas9 are also known and suitable for use in the present invention.

In some embodiments, CRISPR/Cas nuclease-mediated integration utilizes type II CRISPR. Type II CRISPR is one of the most well characterized systems and performs targeted DNA double strand breaks in four consecutive steps. First, two non-coding RNAs, namely a pre-crRNA array and a tracrRNA, are transcribed from the CRISPR locus. Second, the tracrRNA hybridizes to the repeat region of the pre-crRNA and mediates the processing of the pre-crRNA into mature crRNA containing a single spacer sequence. Third, the mature crRNA tracrRNA complex directs Cas9 to target DNA via watson-crick base pairing between a spacer on the crRNA and a pre-spacer on the target DNA adjacent to a pre-spacer adjacent motif (PAM) (an additional requirement for target recognition). Fourth, Cas9 mediates cleavage of the target DNA to create a double strand break within the pre-spacer sequence.

Cas 9-associated CRISPR/Cas system comprises two RNA non-coding components: tracrRNA and pre-crRNA arrays containing nuclease guide sequences (spacers) separated by identical Direct Repeats (DR). To accomplish genome engineering using the CRISPR/Cas system, both functions of these RNAs must be present (see Cong et al, (2013) science xpress 1/10.1126/science 1231143). In some embodiments, the tracrRNA and the pre-crRNA are provided via separate expression constructs or as separate RNAs. In other embodiments, chimeric RNAs are constructed in which an engineered mature crRNA (conferring target specificity) is fused to a tracrRNA (providing interaction with Cas9) to produce a chimeric cr-RNA-tracrRNA hybrid (also referred to as a single guide RNA). (see Jinek supra and Cong supra).

In some embodiments, a single guide RNA containing crRNA and tracrRNA may be engineered to guide Cas9 nuclease to target any desired sequence (e.g., Jinek et al, (2012) Science 337, pages 816-821, Jinek et al, (2013), ehife 2: e00471, David Segal, (2013) ehife 2: e 00563). Thus, the CRISPR/Cas system can be engineered to produce DSBs at the desired target of the genome.

Customized CRISPR/Cas systems are commercially available from, e.g., Dharmacon (Lafayette, CO), and can routinely target and cleave any location of DNA using such customized single-guide RNA sequences. Single-stranded DNA templates for recombination can be synthesized (e.g., via oligonucleotide synthesis methods known in the art and commercially available), or provided in a vector, such as a viral vector, e.g., AAV.

In some embodiments, the cell (e.g., memory B cell or plasmablast) is engineered via TALE nuclease (TALEN) mediated targeted integration of a donor construct (e.g., follistatin donor construct). A "TALE DNA binding domain" or "TALE" is a polypeptide comprising one or more TALE repeat domains/units. The repeat domain is involved in binding of the TALE to its cognate target DNA sequence. A single "repeat unit" (also referred to as a "repeat") is typically 33-35 amino acids in length and exhibits at least some sequence homology to other TALE repeat sequences within a naturally occurring TALE protein. TAL effectors may contain a nuclear localization sequence, an acidic transcription activation domain, and a centralized domain of tandem repeats, where each repeat contains about 34 amino acids that are critical for the DNA binding specificity of these proteins. (e.g., Schornack S, et al (2006) J Plant Physiol 163(3): 256-. TAL effectors depend on sequences comprising about 102bp found in tandem repeats, and the repeats are usually 91-100% homologous to each other (e.g., Bonas et al, (1989) MoI Gen Genet218: 127-136). These DNA binding repeats can be engineered into proteins with novel combinations and numbers of repeats to produce artificial transcription factors that can interact with novel sequences and activate expression of non-endogenous reporters (e.g., Bonas et al, (1989) Mol Gen Genet218: 127-. Engineered TAL proteins can be linked to FokI cleavage half-domains to generate TAL effector domain nuclease fusions (TALENs) to cleave target-specific DNA sequences (e.g., Christian et al, (2010) Genetics epub 10.1534/genetics.110.120717).

Customized TALENs are commercially available from, for example, Thermo Fisher Scientific (Waltham, MA), and can routinely target and cleave any location of DNA.

In some embodiments, the cell (e.g., memory B cell or plasmablast) is engineered via meganuclease-mediated targeted integration of a donor construct (e.g., a follistatin donor construct). Meganucleases (or "homing endonucleases") are endonucleases that bind and cleave double-stranded DNA at recognition sequences greater than 12 base pairs. The naturally occurring meganuclease may be monomeric (e.g., I-SceI) or dimeric (e.g., I-CreI). Naturally occurring meganucleases recognize 15-40 base pair cleavage sites and are generally divided into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cyst box family and the HNH family. Exemplary homing endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII, and I-TevIII. 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-; argast et al (1998) J.mol.biol.280:345-353 and New England Biolabs catalog. The term "meganuclease" includes monomeric meganucleases, dimeric meganucleases, and monomers that associate to form dimeric meganucleases.

In certain embodiments, the methods and compositions described herein utilize nucleases including engineered (non-naturally occurring) homing endonucleases (meganucleases). Recognition sequences for homing endonucleases and meganucleases 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-; argast et al (1998) J.mol.biol.280:345-353 and New England Biolabs catalog. In addition, the DNA binding specificity of homing endonucleases and meganucleases can be engineered to bind to non-natural target sites. See, e.g., Chevalier et al (2002) molecular. cell 10: 895-905; epinat et al (2003) Nucleic Acids Res.31: 2952-2962; ashworth et al (2006) Nature441: 656-659; paques et al (2007) Current Gene Therapy 7: 49-66; U.S. patent publication No. 20070117128. The DNA binding domains of homing endonucleases and meganucleases can be altered in the context of the nuclease as a whole (i.e., such that the nuclease includes a homologous cleavage domain) or can be fused to a heterologous cleavage domain. Custom meganucleases are commercially available from, e.g., New England Biolabs (Ipswich, MA) and can routinely target and cleave any site of DNA.

Engineering of B cells can include, e.g., administering one or more nucleases (e.g., ZFNs, TALENs, CRISPR/Cas, meganucleases) to the B cells via one or more nuclease-encoding vectors such that the B cells uptake the vector comprising the encoded nuclease. The vector may be a viral vector.

In some embodiments, a nuclease cleaves a specific endogenous locus (e.g., a safe harbor gene (safe harbor gene) or a target locus) in a cell (e.g., a memory B cell or a plasma cell) and applies one or more exogenous (donor) sequences (e.g., a transgene) (e.g., one or more vectors comprising these exogenous sequences). In such embodiments, the donor sequence can encode follistatin (e.g., a follistatin transgene). Nucleases can induce double-stranded (DSB) or single-stranded breaks (nicks) in the target DNA. In some embodiments, targeted insertion of a donor transgene (e.g., a follistatin donor transgene) can be performed via Homologous Directed Repair (HDR), non-homologous repair mechanisms (e.g., NHEJ-mediated end capture), or insertion and/or deletion of nucleotides (e.g., endogenous sequences) at the site of integration of the transgene (e.g., 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 one of days 1 through 12 of the in vitro culture. In one embodiment, the cells are electroporated at day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, or day 9 of the in vitro culture. In one embodiment, cells are electroporated at day 2 of in vitro culture to deliver the plasmid.

In one embodiment, the cells are transfected with a transposon. As used herein, the term "transposed" may refer in some embodiments to such a cell transfected with a transposon. A number of 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 system are well known in the art and are suitable for use in the present invention. See, e.g., Hackett P.B. et al, evaluation Risks of institutional Mutagenesis by DNA Transposons in Gene Therapy, Transl Res.2013, 4 months; 161(4) 265-283; hudecek M et al, Going non-visual, the Sleeping Beauty systems break on through to the clinical side, Crit Rev Biochem Mol biol.2017, 8 months; 52(4) 355-380, each of which is incorporated by reference herein in its entirety. In some embodiments, the cells are transfected with sleeping beauty transposons. In some embodiments, the sleeping beauty transposon can be a T2 sleeping beauty transposon or a T4 sleeping beauty transposon. In some embodiments, utilization of the sleeping beauty transposon system can comprise transfecting (e.g., via electroporation) a B cell with a DNA construct encoding the transposon system machinery and a DNA construct encoding the follistatin polypeptide. In some embodiments, the DNA construct encoding the transposon system apparatus may be pCMV-SB100 x. In some embodiments, the use of the sleeping beauty transposon system can comprise transfecting (e.g., via electroporation) a B cell with a DNA construct encoding a follistatin polypeptide, and also transfecting the B cell 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 T2 or T4 sleeping beauty transposon or a Piggybac transposon) at one day of in vitro culture from day 1 to day 12. In one embodiment, the cells are transfected with a transposon (e.g., T2 or T4 sleeping beauty transposon or Piggybac transposon) at day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8 or day 9 of in vitro culture. In one embodiment, the cells are transfected with the minicircle on one of days 1 to 12 of in vitro culture. In one embodiment, cells are transfected with minicircles on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, or day 9 of in vitro culture.

In one embodiment, the cells are transfected with sleeping beauty transposons (e.g., T2 or T4 sleeping beauty transposons) on one of days 1 through 12 in vitro culture. In one embodiment, cells are transduced via electroporation using a sleeping beauty transposon (e.g., T2 or T4) system on day 2 of in vitro culture. In one embodiment, cells are transduced via electroporation using a sleeping beauty transposon (e.g., T2 or T4) system on day 5 of in vitro culture. In one embodiment, cells are transduced via electroporation using a sleeping beauty transposon (e.g., T2 or T4) system on day 8 of in vitro culture. In one embodiment, cells are transduced via electroporation using a sleeping beauty transposon (e.g., T2 or T4) system on day 11 in vitro culture. In one embodiment, cells are transduced via electroporation using a sleeping beauty transposon (e.g., T2 or T4) system on day 14 in vitro culture.

In one embodiment, cells are transfected with Piggybac transposons on one day from day 1 to day 12 of in vitro culture. In one embodiment, cells are transduced via electroporation using the Piggybac transposon at day 2 of in vitro culture. In one embodiment, cells are transduced via electroporation using the Piggybac transposon at day 5 of in vitro culture. In one embodiment, the cells are transduced via electroporation using the Piggybac transposon at day 8 of in vitro culture. In one embodiment, cells are transduced via electroporation using the Piggybac transposon at day 11 of in vitro culture. In one embodiment, cells are transduced via electroporation using the Piggybac transposon at day 14 of in vitro culture.

In one embodiment, the B cell is contacted with a vector comprising a nucleic acid of interest operably linked to a promoter under conditions sufficient to transfect at least a portion of the B cell. In one embodiment, the B cells are contacted with a vector comprising a nucleic acid of interest operably linked to a promoter under conditions sufficient to transfect at least 5% of the B cells. In further embodiments, the B cells are contacted with the vector under conditions sufficient to transfect at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100% of the B cells. In a particular 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 sufficient to transfect at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even 100% of the B cells.

The 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, retrovirus-adenovirus vectors, and vectors derived from Herpes Simplex Virus (HSV), including amplicon vectors, replication-defective HSV, and attenuated HSV (see, e.g., Krisky, Gene Ther.5: 1517-211, 1998; Pfeifer, Annu. Rev. genomics hum. Gene.2: 177-211,2001, each of which is incorporated by reference in its entirety).

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

In one embodiment, B cells are transduced with retroviral vectors using any of a variety of techniques known in the art (see, e.g., Science 12April 1996272: 263-267; Blood 2007,99: 2342-2350; Blood2009,113: 1422-1431; Blood2009, day 10, 8; 114 (15): 3173-80; blood.2003; 101(6): 2167-2174; Current Protocols in Molecular Biology or Current Protocols in Immunology, John ey & Sons, New York, N.Y. (2009)). Additional descriptions of viral transduction of B cells can be found in WO 2011/085247 and WO 2014/152832, each of which is incorporated herein by reference in its entirety.

For example, PBMC, B or T lymphocytes from donors and other B cell cancer cells, such as B-CLL, can be isolated and cultured in IMDM medium or RPMI 1640(GibcoBRL Invitrogen, Auckland, New Zealand) or other suitable medium (serum-free or supplemented with serum (e.g., 5-10% FCS, human AB serum, and serum replacement) and penicillin/streptomycin and/or other suitable supplements as described hereinAgents such as transferrin and/or insulin). In one embodiment, the cells are plated at 1x 10⁵Individual cells were seeded in 48-well plates and concentrated vehicle was added at various doses that one skilled in the art could routinely optimize using routine methods. In one embodiment, B cells were transferred to MS5 cell monolayers in RPMI supplemented with 10% AB serum, 5% FCS, 50ng/ml rhSCF, 10ng/ml rhlL-15, and 5ng/ml rhlL-2, and the media was periodically refreshed as needed. As will be appreciated by those skilled in the art, other suitable media and supplements may be used as desired.

Certain embodiments relate to the use of retroviral vectors or vectors derived from retroviruses. A "retrovirus" is an enveloped RNA virus that is capable of infecting animal cells and utilizing reverse transcriptase early in the infection to produce a copy of DNA from its 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-based retroviral vectors that provide long-term stable expression in target Cells such as hematopoietic precursor Cells and their differentiated progeny (see, e.g., Hawley et al, PNAS USA 93: 10297-.

In one embodiment, a B cell is contacted with a retroviral vector comprising a nucleic acid of interest operably linked to a promoter under conditions sufficient to transduce at least a portion of the B cell. 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 sufficient to transduce at least 2% of the B cells. In further embodiments, the B cells are contacted with the vector under conditions sufficient to transduce at least 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100% resting B cells. In a particular 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 sufficient to transduce at least 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even 100% of the differentiated and activated B cells.

In certain embodiments, prior to transduction, the cells are pre-stimulated with Staphylococcus aureus Cowan (SAC; Calbiochem, San Diego, Calif.) and/or IL-2 at appropriate concentrations known to and routinely optimized by the skilled artisan. Other B cell activating factors (e.g., PMA) as known to the skilled artisan and as described herein may be used.

As noted above, certain embodiments employ lentiviral vectors. The term "lentivirus" refers to a genus of complex retroviruses that are 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, 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 USA96:2988-2993, 1999; Uchida et al, PNAS USA 95: 11939-11944, 1998; Miyoshi et al, Science 283:682-686, 1999; Sutton et al, J Virol 72:5781-5788, 1998; and Frecha et al, blood.112: 4843-52,2008, each of which is incorporated by reference in its entirety).

It has been documented that resting T and B cells can be transduced by VSVG-coated LVs carrying most HIV accessory proteins (vif, vpr, vpu and nef) (see, e.g., Frecha et al, 2010mol. In certain embodiments, the retroviral vector comprises certain minimal sequences from a lentiviral genome, such as an HIV genome or an SIV genome. The lentivirus genome is typically organized into a5 'Long Terminal Repeat (LTR) region, a gag gene, a pol gene, an env gene, auxiliary 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 (repeats) and U5. The U3 region contains enhancer and promoter elements, the U5 region contains a polyadenylation signal, and the R region distinguishes between U3 and U5. The transcript sequences for the R region are present at 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-. Lentiviral vectors may comprise any one or more of these elements of the lentiviral genome to modulate 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 round of infection.

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

In certain embodiments, the viral vector comprises sequences from the 5 'and/or 3' LTRs of a retrovirus, such as a lentivirus. The LTR sequence may be an LTR sequence from any lentivirus of any species. For example, they may be LTR sequences from HIV, SIV, FIV or BIV. Preferably, the LTR sequence is an HIV LTR sequence.

In certain embodiments, the viral vector includes R and U5 sequences from the 5'LTR of lentivirus and an inactivated or "self-inactivated" 3' LTR from lentivirus. A "self-inactivated 3 'LTR" is a 3' Long Terminal Repeat (LTR) that contains a mutation, substitution, or deletion that prevents the LTR sequence from driving expression of a downstream gene. The copy of the U3 region from the 3' LTR serves as a template for the generation of two LTRs in the integrating provirus. Therefore, when the 3' LTR having an inactivated deletion or mutation is integrated into the 5' LTR of the provirus, transcription from the 5' LTR is impossible. This eliminates competition between the viral enhancer/promoter and any internal enhancer/promoter. Self-inactivating 3' LTRs are described, for example, in Zufferey et al, J Virol.72:9873-9880, 1998; miyoshi et al, J Virol.72: 8150-; and Iwakuma et al, J Virology 261: 120-. The self-inactivating 3' LTR may be generated by any method known in the art. In certain embodiments, the U3 element of the 3' LTR contains a deletion of its enhancer sequence, preferably the TATA box, Spl, and/or NF-. kappa.B site. Due to the self-inactivated 3'LTR, the provirus integrated into the host cell genome will contain an inactivated 5' LTR.

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

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

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

Certain embodiments may employ genes encoding one or more selectable markers. Examples include selectable markers useful in eukaryotic or prokaryotic cells, such as genes encoding factors necessary for survival or growth of transformed host cells grown in selective media for drug resistance. Exemplary selection genes encode proteins that confer resistance to antibiotics or other toxins (such as G418, hygromycin B, puromycin, bleomycin, ouabain, blasticidin, ampicillin, neomycin, methotrexate, or tetracycline), complement auxotrophic deficiencies, or supplies can be present on separate plasmids and introduced by co-transfection with viral vectors. In one embodiment, the gene encodes a mutant dihydrofolate reductase (DHFR) that confers resistance to methotrexate. Certain other embodiments may employ genes encoding one or cell surface receptors useful for labeling and detecting or purifying transfected cells, such as the low affinity nerve growth factor receptor (LNGFR) or other such receptors used as transduction tagging systems, see, e.g., Lauer et al, Cancer Gene Ther.2000, 3/7 (3): 430-7.

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

By "functionally related" and "operably linked" is meant, but is not limited to, that the gene (e.g., follistatin gene) is in the correct position and orientation relative to the promoter and/or enhancer such that expression of the gene (e.g., follistatin gene) will be affected when the promoter and/or enhancer is contacted with the appropriate regulatory molecule. Any enhancer/promoter combination that modulates (e.g., increases, decreases) the expression of the viral RNA genome in the packaging cell line, modulates the expression of a selected gene of interest in the infected target cell, or both, can be used.

Promoters are expression control elements formed from DNA sequences that allow polymerase binding and transcription to occur. A promoter is an untranslated sequence located upstream (5') of the initiation codon of a selected target gene (typically within about 100 to 1000 bp) and controls the transcription and translation of the coding polynucleotide sequence to which it is 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 (e.g., a change in temperature). The promoter may be unidirectional or bidirectional. A bidirectional promoter can be used to co-express two genes, such as a gene of interest (e.g., follistatin) and a selectable marker. Alternatively, a bidirectional promoter configuration comprising two promoters in opposite orientations in the same vector may be used, each promoter controlling the expression of a different gene.

As with the methods used to operably link a promoter to a polynucleotide coding sequence, a variety of promoters are known in the art. Both native promoter sequences and a number of heterologous promoters can be used to direct expression of a selected gene of interest. Certain embodiments employ heterologous promoters because they generally allow for higher transcription and higher yields of the desired protein than native promoters.

Certain embodiments may employ heterologous viral promoters. Examples of such promoters include those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus, bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and Simian Virus 40(SV 40). Certain embodiments may employ heterologous mammalian promoters, such as actin promoters, immunoglobulin promoters, heat shock promoters, or promoters associated with native sequences of genes of interest (e.g., follistatin genes). Typically, the promoter is compatible with target cells such as activated B lymphocytes, plasma B cells, memory B cells, or other lymphocyte target cells.

Certain embodiments may employ one or more of RNA polymerase II and III promoters. Suitable choices for RNA polymerase III promoters can be found, for example, in Paule and white. nucleic Acids research, volume 28, pp1283-1298,2000, which are incorporated by reference in their entirety. RNA polymerase II and III promoters also include any synthetic or engineered DNA segment that can direct transcription of its downstream RNA coding sequence by RNA polymerase II or III, respectively. In addition, the RNA polymerase II or III (Pol II or III) promoter used as part of the viral vector may be inducible. Any suitable inducible Pol II or III promoter may be used with the methods described herein. Exemplary Pol II or III promoters include Ohkawa and Taira, Human Gene Therapy, Vol.11, pp577-585, 2000; and the tetracycline-responsive promoters provided in Meissner et al, Nucleic Acids Research, Vol.29, pp1672-1682,2001, each of which is incorporated by reference in its entirety.

Non-limiting examples of constitutive promoters that can be used include ubiquitin promoters, CMV promoters (see, e.g., Karasuyama et al, J.Exp.Med.169:13,1989), beta-actin (see, e.g., Gunning et al, PNAS USA 84:4831-4835,1987), elongation factor-1 alpha (EF-1 alpha) promoters, CAG promoters, and pgk promoters (see, e.g., Adra et al, Gene 60:65-74, 1987); Singer-Sam et al, Gene 32: 409-; and Dobson et al, Nucleic Acids Res.10:2635-2637,1982, each of which is 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 myoblast promoter (Yee et al, Genes and Development 7:1277-1289.1993), and the thyyl promoter (see, e.g., Gundersen et al, Gene 113: 207-214, 1992).

Additional examples of promoters include the ubiquitin-C promoter, the human mu heavy chain promoter or the Ig heavy chain promoter (e.g., MH), and the human kappa light chain promoter or the Ig light chain promoter (e.g., EEK), which are functional in B-lymphocytes. The MH promoter contains the human mu heavy chain promoter followed by the iE mu enhancer flanked by matrix binding regions, and the EEK promoter contains the kappa light chain promoter followed by the intron enhancer (iE κ), matrix binding region, and 3' enhancer (3E κ) (see, e.g., Luo et al, blood.113: 1422) -1431,2009, and U.S. patent application publication No. 2010/0203630). Thus, certain embodiments may employ one or more of these promoter or enhancer elements.

In one embodiment, one promoter drives expression of the 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 the production of a selectable marker (e.g., DHFR) and the mini-CAG promoter (see, e.g., Fan et al Human Gene Therapy 10: 2273-2285, 1999) drives the expression of a Gene of interest (e.g., follistatin).

As described above, certain embodiments employ enhancer elements, such as internal enhancers, to increase the expression of the target gene. Enhancers are cis-acting elements of DNA, usually about 10 to 300bp in length, that act on a promoter to increase its transcription. Enhancer sequences can be derived from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, insulin), such as the iota μ enhancer, the iota intron enhancer, and the 3' kappa enhancer. Also included are enhancers from eukaryotic viruses, including the SV40 enhancer on the posterior side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the posterior side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at the 5' or 3' position of the antigen specific polynucleotide sequence, but is preferably located at the 5' site of the promoter. One skilled in the art will select the appropriate enhancer based on the desired expression pattern.

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

Certain viral vectors contain cis-acting packaging sequences to facilitate the incorporation of genomic viral RNA into the viral particle. Examples include psi sequences. Such cis-acting sequences are known in the art. In certain embodiments, the viral vectors described herein can express two or more genes, which can be achieved, for example, by incorporating an internal promoter operably linked to each individual gene other than the first gene, by incorporating elements that promote co-expression such as Internal Ribosome Entry Sequence (IRES) elements (U.S. Pat. No. 4,937,190, incorporated by introduction) or 2A elements, or both. By way of illustration only, an IRES or 2A element may 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 a heavy or light chain) may be located immediately downstream of a promoter, and a second coding region (encoding another chain) may be located downstream of the first coding region, with an IRES or 2A element located between the first and second coding regions, preferably immediately before 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, IRES elements of encephalomyelitis Virus (EMCV), Foot and Mouth Disease Virus (FMDV), Treell encephalomyelitis Virus (TMEV), Human Rhinovirus (HRV), coxsackie Virus (CSV), Poliovirus (POLIO), Hepatitis A Virus (HAV), Hepatitis C Virus (HCV), and pestiviruses (e.g., hog cholera lentogen (HOCV) and Bovine Viral Diarrhea Virus (BVDV)) (see, e.g., Le et al, Virus Genes 12: 135-. One example of a 2A element includes the F2A sequence from foot-and-mouth disease virus.

In certain embodiments, the vectors provided herein further contain additional genetic elements to achieve the desired results. For example, certain viral vectors can include signals that facilitate entry of the nucleus of the viral genome into a target cell, such as the HIV-1 flap signal. As a further example, certain viral vectors can include elements that facilitate characterization of the proviral integration site in the target cell, such as tRNA amber inhibitor sequences. Certain viral vectors may contain one or more genetic elements designed to enhance expression of a gene of interest (e.g., a follistatin gene). For example, woodchuck hepatitis virus response elements (WREs) can be placed in constructs (see, e.g., Zufferey et al, J.Virol.74: 3668-. As another example, chicken beta-globin spacers may also be included in the construct. It has been shown that this element reduces the possibility of silencing integrated DNA in the target cell due to methylation and heterochromatin. In addition, the insulator can protect internal enhancers, promoters and foreign genes from positive or negative positional effects of surrounding DNA at integration sites on the chromosome. Certain embodiments employ each of these genetic elements. In another embodiment, The viral vectors provided herein may also contain Ubiquitous Chromatin Opening Elements (UCOEs) to increase expression (see, e.g., Zhang F et al, Molecular Therapy: The journal of The American Society of Gene Therapy 9/2010; 18(9): 1640-9.).

In certain embodiments, a viral vector (e.g., retrovirus, lentivirus) provided herein is "pseudotyped" with one or more selected viral glycoproteins or envelope proteins that are targeted primarily to a selected cell type. Pseudotyping (pseudo-typing) generally refers to the incorporation of one or more heterologous viral glycoproteins into a cell surface viral particle, typically allowing the viral particle to infect selected cells other than its normal target cell. A "heterologous" element is derived from a virus other than the virus from which the RNA genome of the viral vector is derived. Typically, the glycoprotein-encoding region of a viral vector has been genetically altered, such as by deletion, to prevent expression of its own glycoprotein. By way of illustration only, the envelope glycoproteins gp41 and/or gp120 of an HIV-derived lentiviral vector are typically deleted prior to pseudotyping with a heterologous viral glycoprotein.

In certain embodiments, the viral vector is pseudotyped with a heterologous viral glycoprotein that targets B lymphocytes. In certain embodiments, the viral glycoprotein allows for selective infection or transduction of resting or quiescent B lymphocytes. In certain embodiments, the viral glycoprotein allows selective infection of B lymphocyte plasma cells, plasmablasts, and activated B cells. In certain embodiments, the viral glycoprotein allows infection or transduction of quiescent B lymphocytes, plasmablasts, plasma cells, and activated B cells. In certain embodiments, the viral glycoprotein allows infection of B-cell chronic lymphocytic leukemia cells. In one embodiment, the viral vector is pseudotyped with VSV-G. In another embodiment, the heterologous viral glycoprotein is derived from a glycoprotein of a measles virus (e.g., an Edmonton measles virus). Certain embodiments pseudotyping measles virus glycoprotein hemagglutinin (H), fusion protein (F), or both (see, e.g., Frecha et al, blood.112: 4843-. In one embodiment, the viral vector is pseudotyped with Gibbon Ape Leukemia Virus (GALV). In one embodiment, the viral vector is pseudotyped with feline endogenous retrovirus (RD 114). In one embodiment, the viral vector is pseudotyped with baboon endogenous retrovirus (BaEV). In one embodiment, the viral vector is pseudotyped with Murine Leukemia Virus (MLV). In one embodiment, the viral vector is pseudotyped with Gibbon Ape Leukemia Virus (GALV). In further embodiments, the viral vector includes an embedded antibody binding domain, such as one or more variable regions (e.g., heavy and light chain variable regions), for targeting the vector to a particular cell type.

The generation of viral vectors can be accomplished using any suitable genetic engineering technique known in the art, including, but not limited to, standard techniques of restriction endonuclease digestion, ligation, transformation, plasmid purification, PCR amplification and DNA sequencing, such as, for example, Sambrook et al (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y. (1989)); coffin et al (retroviruses. Cold Spring Harbor Laboratory Press, N.Y. (1997)) and "RNA Viruses: A Practical Approach" (Alan J. Cann, eds., Oxford University Press, (2000).

Various methods known in the art can be used to generate suitable retroviral particles whose genome comprises an RNA copy of the viral vector. As one approach, the viral vector may be introduced into a packaging cell line that packages viral genomic RNA based on the viral vector into viral particles having the desired target cell specificity. Packaging cell lines typically provide viral proteins, including the structural gag protein, the enzymatic pol protein, and the envelope glycoproteins, in trans, which are required to package the viral genomic RNA into the viral particle and infect the target cell.

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

Therapeutic agents

As used herein, a "gene of interest" or "gene" or "nucleic acid of interest" refers to a transgene to be expressed in a cell transfected with a target. In a particular embodiment, the gene is a follistatin gene. Although the term "gene" may be used, this does not mean that this is a gene present in genomic DNA and may be used interchangeably with the term "nucleic acid". Typically, the nucleic acid of interest provides a suitable nucleic acid for encoding a therapeutic agent (e.g., follistatin) and may comprise cDNA or DNA, and may or may not comprise an intron, but typically does not comprise an intron. As noted elsewhere, the nucleic acid of interest is operably linked to an expression control sequence to efficiently express the protein of interest in the target cell. In certain embodiments, the vectors described herein may comprise one or more genes of interest, and may include 2, 3, 4, or 5 or more genes of interest, such as, for example, the heavy and light chains of immunoglobulins that may be organized using internal promoters as described herein.

As used herein, the recitation "polynucleotide" or "nucleic acid" means mRNA, RNA, cRNA, cDNA, or DNA. The term generally refers to a polymeric form of nucleotides (ribonucleotides or deoxynucleotides or modified forms of either type of nucleotide) that are at least 10 bases in length. The term includes single-and double-stranded forms of DNA and RNA. The target nucleic acid or gene may be any nucleic acid encoding a protein of interest.

In some embodiments, any one of the embodiments disclosed herein may utilize a gene of interest that is a follistatin protein. In some embodiments, the follistatin protein can be any one of the follistatin proteins shown in SEQ ID NOs: 1-4. Thus, in some embodiments, a therapeutic agent delivered to a genetically modified B cell as described herein can be a follistatin protein.

Follistatin

In various aspects, the disclosure relates to B cells engineered to express follistatin (e.g., one or more follistatin polypeptides). As used herein, the term "follistatin" refers to a family of Follistatin (FST) and follistatin-related proteins derived from any species. Follistatin is an autocrine glycoprotein expressed in almost all tissues of higher animals. It was originally isolated from follicular fluid and was identified as the portion of the protein that inhibits the secretion of Follicle Stimulating Hormone (FSH) from the anterior pituitary, and is therefore referred to as FSH-inhibiting protein (FSP). Subsequently, it has been determined that its primary function is the binding and neutralization of members of the TGF-. beta.superfamily, including, for example, activin, a paracrine hormone that enhances FSH secretion in the anterior pituitary.

In some embodiments, the term "follistatin polypeptide" or "follistatin" is used to refer to any naturally occurring polypeptide comprising the follistatin family and which retains useful activity, including, for example, polypeptides that are ligand-bound (e.g., myosin, GDF-11, activin a, activin B) or any variant of heparin-binding (including mutants, fragments, fusions and peptidomimetic (peptidomimetic) forms). For example, in some embodiments, a follistatin polypeptide can include a polypeptide that comprises an amino acid sequence derived from any known follistatin sequence that is at least about 80% identical, and preferably at least 85%, 90%, 95%, 97%, 99% or more identical to the sequence of the follistatin polypeptide.

Follistatin is a single-chain polypeptide with a molecular weight range of 31kDa to 49kDa based on alternative mRNA splicing and alternative glycosylation of proteins. The human gene encoding Follistatin (FST) has 6 exons spanning 5329bp on chromosome 5q11.2 and produces two major transcripts: transcript variant FST344(1122bp) and transcript FST317(1386 bp). Exon 1 in FST encodes the follistatin signal peptide, exon 2 encodes the follistatin N-terminal domain, and each of exons 3-5 encodes a follistatin module. Either exon 6A (which encodes the acidic region in FST344) or exon 6B (which contains two bases in the stop codon of FST317) was used for alternative splicing (Shimasaki, S et al, 1988).

These alternatively spliced mrnas (FST344 and FST317) result in 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 into follistatin 303(FST 303). Analysis of the amino acid sequence has revealed that the native human follistatin polypeptide comprises 5 domains (starting from the N-terminal side): signal sequence peptides (amino acids 1-29 of SEQ ID NO: 1), 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, U.S. A.,1988, Vol.85, No. 12, pp 4218-.

The human follistatin-288 (FST288) precursor (i.e., FST317) has the following amino acid sequence, where the signal peptide is shown in bold, the N-terminal domain (FSN) is shown by single underlining, and follistatin domains I-III (FSI, FSII, FSIII) are shown by double underlining.

The processed (mature) human follistatin variant (FST288) has the following amino acid sequence, wherein the N-terminal domain is indicated by single underlining and follistatin domains I-III are indicated by double underlining. Furthermore, it is understood that any initial amino acid G or N preceding the first cysteine may be removed by processing or intentionally removed without any consequence and also includes polypeptides comprising such slightly smaller polypeptides.

The human follistatin-315 (FST315) precursor (i.e., FST344) has an amino acid sequence in which the signal peptide is in bold, the N-terminal domain (FSN) is single underlined, and follistatin domains I-III (FSI, FSII, FSIII) are double underlined (NCBI accession No. AAH 04107.1; 344 amino acids).

Processed (mature) human FST315 has the following amino acid sequence, wherein the N-terminal domain is indicated by single underlining and the follistatin domains I-III are indicated by double underlining. Furthermore, it is understood that any initial amino acid G or N preceding the first cysteine may be removed by processing or intentionally removed without any consequence and also includes polypeptides comprising such slightly smaller polypeptides.

Follistatin polypeptides of the disclosure may include any naturally occurring domain of a follistatin protein as well as variants thereof (e.g., mutants, fragments, and peptidomimetic forms) that retain useful activity. For example, it is well known that FST315 and FST288 have high affinity for both activin (activin a and activin B) and myostatin (and the closely related GDF11), and that follistatin domains (e.g., FSN and FSD I-III) are thought to be involved in binding of this TGF- β ligand. However, it is believed that each of these three domains may have different affinities for these TGF- β ligands. For example, studies have demonstrated that polypeptide constructs comprising only The N-terminal domain (FSN) and two FSDI domains in tandem retain high affinity for myostatin, show little or no affinity for activin and promote systemic muscle growth when introduced into mice by gene expression (Nakatani et al, The FASEB Journal, vol.22477-487 (2008)).

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

Accordingly, the present disclosure provides polynucleotides (isolated or purified or pure polynucleotides) encoding the therapeutic agents of the disclosure (e.g., follistatin) for use in genetically modified B cells, vectors (including cloning vectors and expression vectors) comprising such polynucleotides, and cells (e.g., host cells) transformed or transfected with polynucleotides or vectors according to the disclosure. In certain embodiments, any of the embodiments disclosed in the present disclosure may utilize a follistatin (e.g., for expression in B cells) selected from the follistatin polypeptides of SEQ ID NOs 1-4. In certain embodiments, any of the embodiments disclosed in the present disclosure may utilize follistatin (e.g., for expression in B cells) as a human follistatin FST344 splice site variant. In certain embodiments, polynucleotides (DNA or RNA) encoding a protein of interest of the present disclosure (e.g., follistatin) are contemplated. Also contemplated herein are expression cassettes encoding the proteins of interest.

The disclosure also relates to vectors comprising the polynucleotides of the disclosure and in particular to recombinant expression constructs. In one embodiment, the present disclosure contemplates vectors comprising polynucleotides encoding proteins of the disclosure (e.g., follistatin) and polynucleotide sequences that cause or facilitate transcription, translation, and processing of such protein coding 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, which may be based on plasmids, phagemids (phasmids), cosmids, viruses, artificial chromosomes, or any nucleic acid vehicle known in the art suitable for amplifying, transferring, and/or expressing the polynucleotides contained therein.

As used herein, unless otherwise described with respect to viral vectors, "vector" means a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Exemplary vectors include plasmids, minicircles, transposons (e.g., sleeping beauty transposons), yeast artificial chromosomes, self-replicating RNAs, and viral genomes. Some vectors may replicate autonomously in the host cell, whereas other vectors may integrate into the genome of the host cell and thereby replicate together with the host genome. In addition, certain vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors") that contain nucleic acid sequences operably linked to expression control sequences and are therefore capable of directing the expression of those sequences. In certain embodiments, the expression construct is derived from a plasmid vector. Illustrative constructs include a modified pNASS vector (Clontech, Palo Alto, Calif.) having a nucleic acid sequence encoding an ampicillin resistance gene, a polyadenylation signal and a T7 promoter site; pDEF38 and pNEF38(CMC ICOS biologices, Inc.), which has the CHEF1 promoter; and pD18(Lonza), which has a CMV promoter. Other suitable mammalian expression vectors are also well known (see, e.g., Ausubel et al, 1995; Sambrook et al, supra; see also, e.g., catalogues from Invitrogen, San Diego, CA; Novagen, Madison, Wl; Pharmacia, Piscataway, NJ).

Useful constructs can be prepared which include the dihydrofolate reductase (DHFR) coding sequence under appropriate regulatory control to facilitate enhanced levels of fusion protein production resulting from gene amplification following application of an appropriate selection agent (e.g., methotrexate). In one embodiment, successful transposed B cells are enriched for using bifunctional transposons encoding therapeutic genes (e.g., FST) and drug-resistant DHFR in conjunction with incubation in Methotrexate (MTX), resulting in a more efficient product.

In general, a recombinant expression vector will include an origin of replication and a selectable marker that permits transformation of the host cell, as well as a promoter derived from a highly expressed gene to direct transcription of downstream structural sequences, as described above. Vectors operably linked to polynucleotides according to the present disclosure produce cloning or expression constructs. Exemplary cloning/expression recombinants contain at least one expression control element, such as a promoter, operably linked to a polynucleotide of the present disclosure. Additional expression control elements, such as enhancers, factor-specific binding sites, terminators, and ribosome binding sites, are also contemplated in vectors and cloning/expression constructs according to the present disclosure. Heterologous structural sequences of polynucleotides according to the present disclosure are assembled at appropriate stages with translation initiation and termination sequences. Thus, for example, the encoding nucleic acids provided herein can be included in any of a variety of expression vector constructs (e.g., minicircles) as recombinant expression constructs for expressing such proteins in a host cell.

The appropriate DNA sequence may be inserted into the vector, for example, by a variety of procedures. Typically, the DNA sequence is inserted into the appropriate restriction endonuclease cleavage site by procedures known in the art. Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligases, DNA polymerases, restriction endonucleases and the like, and various isolation techniques are contemplated. Various standard techniques are described, for example, in Ausubel et al (Current Protocols in Molecular Biology, Greene Publ.Assoc.Inc. & John Wiley & Sons, Inc., Boston, MA, 1993); sambrook et al (Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, NY, 1989); maniatis et al (Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, NY, 1982); glover (editor) (DNA Cloning Vol.I and II, IRL Press, Oxford, UK, 1985); hames and Higgins (editors) (Nucleic Acid Hybridization, IRL Press, Oxford, UK, 1985); and elsewhere.

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 regulatory promoter) to direct mRNA synthesis. Representative examples of such expression control sequences include promoters of eukaryotic cells or viruses thereof, as described above. The promoter region can be selected from any desired gene using CAT (chloramphenicol transferase) vector, kanamycin vector, or other vector with selectable markers. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTR from retroviruses, and mouse metallothionein-1. Selection of appropriate vectors and promoters is well within the level of ordinary skill in the art, and the preparation of certain particularly preferred recombinant expression constructs including at least one promoter or regulatory promoter operably linked to a nucleic acid encoding a protein or polypeptide according to the present disclosure is described herein.

Variants of the polynucleotides of the disclosure are also contemplated. A polynucleotide variant is 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 hybridizes to one of those polynucleotides of a defined sequence under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate at about 65-68 ℃ or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 42 ℃. Polynucleotide variants retain the ability to encode a binding domain or fusion protein thereof having the functionality described herein.

The term "stringent" is used to refer to conditions that are commonly understood in the art as stringent. Hybridization stringency is primarily determined by temperature, ionic strength, and the concentration of denaturing agents (e.g., formamide). Examples of stringent conditions for hybridization and washing are 0.015M sodium chloride, 0.0015M sodium citrate at about 65-68 ℃ or 0.015M sodium chloride, 0.0015M sodium citrate and 50% formamide at about 42 ℃ (see Sambrook et al, Molecular Cloning: A Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). More stringent conditions (e.g., higher temperature, lower ionic strength, higher formamide or other denaturant) may also be used; however, the rate of hybridization will be affected. In cases where deoxyoligonucleotide hybridization is involved, additional exemplary stringent hybridization conditions include washing in 6x SSC, 0.05% sodium pyrophosphate at 37 ℃ (for 14 base oligonucleotides), 48 ℃ (for 17 base oligonucleotides), 55 ℃ (for 20 base oligonucleotides), and 60 ℃ (for 23 base oligonucleotides).

Another aspect of the disclosure provides a host cell transformed or transfected with or otherwise containing any of the polynucleotides or vector/expression constructs (e.g., follistatin polynucleotides or vector/expression constructs) of the disclosure. The polynucleotides or cloning/expression constructs of the present disclosure are introduced into a suitable cell using any method known in the art, including transformation, transfection, and transduction (e.g., any of the methods disclosed herein). Host cells include cells of a subject undergoing ex vivo cell therapy, including ex vivo gene therapy. Eukaryotic host cells contemplated as being an aspect of the present disclosure when carrying a polynucleotide, vector or protein according to the present disclosure include, in addition to the subject's own cells (e.g., 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. 2003/0115614), COS cells (e.g., COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, a549, PC12, K562, HEK293 cells, HepG2 cells, N cells, 3T3 cells, spodoptera frugiperda cells (e.g., Sf9 cells), saccharomyces cerevisiae cells, and any other eukaryotic cell known in the art to be useful for expressing and optionally isolating a protein or peptide according to the present disclosure. Prokaryotic cells are also contemplated, including Escherichia coli, Bacillus subtilis, Salmonella typhimurium, Streptomyces, or any prokaryotic cell known in the art suitable for expression and optional isolation of a protein or peptide according to the present disclosure. In isolating proteins or peptides from prokaryotic cells, it is specifically contemplated that techniques known in the art for extracting proteins from inclusion bodies can be used. The selection of an appropriate host is within the purview of one skilled in the art in light of the teachings herein. Host cells that glycosylate the fusion proteins of the present disclosure are contemplated.

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

Cells and compositions

In one embodiment, the modified B cells described herein have been activated/differentiated and transfected in vitro to express a therapeutic agent (e.g., follistatin) as described herein. In one embodiment, the modified B cells described herein have been activated/differentiated and engineered in vitro (e.g., using targeted transgene integration methods such as zinc finger nuclease, TALEN, meganuclease, or CRISPR/CAS 9-mediated transgene integration) to express a therapeutic agent (e.g., follistatin) as described herein. In one embodiment, the composition comprises B cells that have differentiated into plasma B cells, have been transfected or otherwise engineered, and express one or more proteins of interest (e.g., follistatin). The target cell population, such as the transfected or otherwise engineered and activated B cell population of the present disclosure, can be administered alone or in a pharmaceutical composition in cooperation with diluents and/or other components such as cytokines or cell populations.

In one embodiment, modified B cells that have been engineered to express one or more proteins of interest (e.g., follistatin) are harvested from culture after in vitro activation/differentiation at a time point where the modified B cells have optimal migratory capacity for a particular chemoattractant. In some embodiments, the optimal migratory capacity can be at day 7, day 8, or day 9 of B cell culture. In some embodiments, the optimal migratory capacity can be at day 5, day 6, or day 7 of B cell culture after transfection or engineering. In some embodiments, the optimal migratory capacity can be at or after day 8 of B cell culture after transfection or engineering (e.g., day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, or later than day 20). In some embodiments, the optimal migratory capacity may be before day 10 of B cell culture. In some embodiments, the optimal migratory capacity may be before day 8 of B cell culture after transfection or engineering. In some embodiments, the optimal migratory capacity can be at day 6 or day 7 of B cell culture. In some embodiments, optimal migratory capacity can be at day 4 or day 5 of B cell culture after transfection or engineering. In some embodiments, the optimal migratory capacity may be before day 9 of B cell culture. In some embodiments, the optimal migratory capacity can be prior to day 7 of B cell culture after transfection or engineering. In some embodiments, optimal migratory capacity is optimal for modified B cells that home to CXCL 12. In some embodiments, the optimal migratory capacity is optimal for modified B cells that home to the bone marrow of a subject receiving one or more administrations of modified B cells. In some embodiments, the B cells are harvested for administration to the subject at about day 7 to about day 9 of culture for optimal migratory capacity of CXCL12 and/or the subject's bone marrow. In some embodiments, B cells are harvested for administration to a subject at about day 5 to about day 7 of the culture after transfection or engineering, at an optimal migratory capacity of CXCL12 and/or the subject's bone marrow. In some embodiments, the B cells are harvested for administration to the subject at up to CXCL12 and/or optimal migratory capacity of the subject's bone marrow before about day 10 of culture. In some embodiments, B cells are harvested for administration to a subject at up to the optimal migratory capacity of CXCL12 and/or the subject's bone marrow prior to about day 8 of culture after transfection or engineering. In some embodiments, optimal migratory capacity is optimal for modified B cells that home to CXCL 13. In some embodiments, the optimal migratory capacity is optimal for modified B cells that home to a site of inflammation in a subject receiving one or more administrations of the modified B cells. In some embodiments, the B cells are harvested for administration to the subject on about day 6 or about day 7 of culture to achieve optimal migratory capacity for CXCL13 and/or a site of inflammation in the subject. In some embodiments, B cells are harvested for administration to a subject at about day 4 or about day 5 of culture after transfection or engineering, for optimal migratory capacity to CXCL13 and/or a site of inflammation in the subject. In some embodiments, B cells are harvested for administration to a subject prior to about day 10 in culture for optimal migratory capacity to CXCL13 and/or a site of inflammation. In some embodiments, B cells are harvested for administration to a subject at optimal migratory capacity to CXCL13 and/or sites of inflammation prior to about day 8 of culture after transfection or engineering.

In some embodiments, optimal migratory capacity is optimal for modified B cells that home to CXCL12 and CXCL 13. In some embodiments, B cells are harvested on day 7 of B cell culture with optimal migratory capacity for homing to CXCL12 and CXCL 13. In some embodiments, B cells are harvested at day 5 of B cell culture after transfection or engineering with optimal migratory capacity to home to CXCL12 and CXCL 13.

In some embodiments, the engineered B cells are harvested when at least about 20% of the B cells migrate to a particular chemotaxis in the chemotaxis assay. For example, and not by way of example, engineered B cells (e.g., FST-producing B cells) can be harvested when at least about 20% of the B cells migrate to CXCL12 in a chemotaxis assay. Alternatively, in another non-limiting example, engineered B cells (e.g., FST-producing B cells) can be harvested when at least about 20% of the B cells migrate to CXCL13 in a chemotaxis assay. In addition, engineered B cells (e.g., FST-producing B cells) can be harvested when at least about 30% of the B cells migrate to a particular chemotaxis assay (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 to a particular chemotaxis assay (e.g., CXCL12 or CXCL 13). In addition, engineered B cells (e.g., IDUA-producing B cells) can be harvested when greater than 70% of the B cells migrate in the chemotaxis assay. Such chemotaxis assays are known in the art.

Briefly, the cell compositions of the present disclosure may comprise a differentiated and activated B cell population that has been transfected and expresses a therapeutic agent (e.g., follistatin) as described herein, 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 dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents, such as EDTA or glutathione; adjuvants (such as aluminum hydroxide); and a preservative. The compositions of the present disclosure are preferably formulated for intravenous or subcutaneous administration.

In one embodiment, the purity of the cell composition is assessed prior to administration. In another embodiment, the cell composition is tested for robustness of therapeutic agent production. In one embodiment, the cell composition is tested for sterility. In another embodiment, the cell composition is screened to confirm that it matches the recipient subject.

In one embodiment, the engineered B cell population is assessed for polyclonality prior to administration to a subject. In some embodiments, ensuring the polyclonality of the final cell product is an important safety parameter. In particular, the emergence of dominant clones may be considered to be likely to lead to tumorigenesis or autoimmune disease in vivo. The polyclonality may be assessed by any means known in the art or described herein. For example, in some embodiments, the polyclonality is assessed by sequencing (e.g., by deep sequencing) B cell receptors expressed in the engineered B cell population. Since the B cell receptor undergoes changes during B cell development, making it unique between B cells, this method allows quantification of how many cells share the same B cell receptor sequence (meaning they are cloned). Thus, in some embodiments, the more B cells in an engineered B cell population that express the same B cell receptor sequence, the more clones of the population and, therefore, the less safe to administer the population to a subject. Conversely, in some embodiments, the fewer B cells in the engineered B cell population that express the same B cell receptor sequence, the fewer clones of the population (i.e., more polyclonal), and thus the greater the safety of administering the population to a subject.

In some embodiments, the engineered B cells are administered to a subject after they have been determined to have sufficient polyclonality. For example, engineered B cells can be administered to a subject after determining that no particular B cell clone in the final population accounts for more than about 0.2% of the total B cell population. The engineered B cells can be administered to the subject after determining that no particular B cell clone in the final population accounts for 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 particular embodiments, engineered B cells (e.g., which produce follistatin) can be administered to a subject after determining that no particular B cell clone in the final population accounts for more than about 0.03% of the total B cell population.

In one embodiment, the cell composition is stored and/or transported at 4 ℃. In another embodiment, the cell composition is frozen for storage and/or transport. The cell composition may be frozen at, for example, -20 ℃ or-80 ℃. In one embodiment, the step of freezing the cell composition comprises liquid nitrogen. In one embodiment, the cell composition is frozen using a controlled rate freezer. Thus, the methods described herein may further comprise a thawing step.

Application method

One aspect of the invention relates to the in vivo delivery of a therapeutic agent (e.g., follistatin) via the delivery of modified B cells engineered to express the therapeutic agent (e.g., follistatin). In particular embodiments, B cells modified to express follistatin are used in methods of treating and/or preventing chronic diseases and disorders and/or methods for increasing muscle size or strength in a patient.

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

Although the appropriate dosage can be determined by clinical trials, the number and frequency of administrations will be determined by factors such as the condition of the patient and the type and severity of the patient's disease.

In one embodiment, a single dose of modified B cells is administered to a subject. In one embodiment, two or more doses of modified B cells are administered sequentially to a subject. In one embodiment, three doses of modified B cells are administered sequentially to the subject. In one embodiment, a dose of modified B cells is administered to a subject once a week, once every two weeks, once a month, once every two months, once a quarter, once every half year (semiannual), once a year, or once every two years (biannualy). In one embodiment, a second or subsequent dose of modified B cells is administered to the subject when the amount of therapeutic agent produced by the modified B cells is decreased.

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

When an "effective amount" or a "therapeutic amount" is indicated, the precise amount of the composition of the present disclosure to be administered can be determined by a physician considering individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). The B cell composition may also be administered in multiple doses as appropriate. The cells can be administered by using infusion techniques commonly known in immunotherapy (see, e.g., Rosenberg et al, New Eng.J.of Med.319:1676,1988).

Optimal dosages and treatment regimens for a particular patient may be determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly. The treatment can also be adjusted after measuring the level of the therapeutic agent (e.g., follistatin) in the biological sample (e.g., a bodily fluid, such as plasma or tissue sample), which can also be used to assess treatment efficacy, and can be adjusted accordingly to increase or decrease treatment.

In some aspects of the disclosure, the optimal dose of modified B cells for use in a multi-dose regimen may be determined by: the method includes the steps of first determining an optimal single dose concentration of B cells for the subject, reducing the number of B cells present in the optimal single dose concentration to provide a sub-optimal single dose concentration of modified B cells, and administering to the subject the modified B cells at the sub-optimal single dose concentration of two or more doses. In some aspects, a sub-optimal single dose concentration of 2, 3, or more doses of modified B cells is administered to the subject. In some aspects, administering 2, 3, or more doses of a suboptimal single dose concentration of modified B cells to a subject results in the synergistic production of a therapeutic polypeptide that the modified B cells are engineered to express in vivo. In some aspects, the sub-optimal single dose concentration is 1/2 or 3, 4, 5,6, 7, 8, 9, 10 times the optimal single dose concentration, or less than the optimal single dose concentration. In some aspects, the therapeutic polypeptide is follistatin.

In some aspects of the disclosure, a lower amount of 10 may be administered⁶In the kilogram range (10 per patient)⁶-10¹¹) Transfected B cells of the disclosure. In certain embodiments, the B cells are administered at 1x 10⁴、5x 10⁴、1x 10⁵、5x 10⁵、1x 10⁶、5x 10⁶、1x 10⁷、5x 10⁷、1x 10⁸、5x 10⁸、5x 10⁹、1x 10¹⁰、5x 10¹⁰、1x 10¹¹、5x 10¹¹Or 1x 10¹²The individual cells are administered to a subject. The B cell composition may be administered multiple times at doses within these ranges. The cells may be autologous or heterologous (e.g., allogeneic) to the patient being treated. If desired, treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines, and/or chemokines described herein (e.g., GM-CSF, IL-4, IL-6, IL-13, IL-21, Flt3-L, RANTES, MIP1 α, BAFF, etc.) to enhance induction of an immune response and engraftment of infused B cells.

Administration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, infusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intrathecally, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. The compositions described herein may be administered directly to a patient into the nervous system. In one embodiment, the B cell composition of the present disclosure is administered to a patient by intradermal or subcutaneous injection. In another embodiment, a B cell composition as described herein is preferably administered by i.v. injection. The composition of B cells may be injected directly into the tumor, lymph node, bone marrow or site of infection.

In yet another embodiment, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer,1990, Science249: 1527-. In another embodiment, polymeric materials may 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 yet another embodiment, a Controlled Release system can be placed in proximity to the therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Medical Applications of Controlled Release,1984, Langer and Wise (ed.), CRC pres, Boca Raton, fla., vol.2, pp. 115-138).

The B cell compositions of the present disclosure can also be administered using any number of matrices. In the context of Tissue Engineering, matrices have been used for many years (see, e.g., Principles of Tissue Engineering (Lanza, Langer and Chick (ed)), 1997. the present disclosure utilizes such matrices in a new context for the support and maintenance of B cells for artificial lymphoid organs accordingly, the present disclosure may utilize those matrix compositions and formulations that have proven useful in Tissue Engineering In the case of an in vivo, the matrix is not biodegradable, such as an implant; or may be biodegradable. The matrix may take the form of a sponge, implant, tube, telfa pad, fiber, hollow fiber, lyophilized component, gel, powder, porous composition, or nanoparticle. In addition, the matrix may be designed to allow for sustained release of seeded cells or produced cytokines or other active agents. In certain embodiments, the matrices of the present disclosure are flexible and resilient and can be described as semi-solid scaffolds that are permeable to substances such as inorganic salts, aqueous fluids, and dissolved gaseous substances including oxygen.

Matrices are used herein as examples of biocompatible materials. However, the present disclosure is not limited to a substrate, and thus, wherever the term substrate or substrates appears, these terms should be understood to include devices and other substances that allow cellular retention or cell traversal, are biocompatible, and are capable of allowing macromolecules to traverse directly through a substance, such that the substance itself is a semi-permeable membrane, or is used in conjunction with a particular semi-permeable substance.

In certain embodiments of the present disclosure, B cells transfected and activated using the methods described herein or other methods known in the art are administered to a patient in conjunction with (e.g., before, simultaneously with, or after) a number of related therapeutic modalities, including but not limited to treatment with agents such as antiviral agents, chemotherapy, radiation, immunosuppressive agents such as cyclosporine, bisufin, bortezomib, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunoablative agents (immunological agents) such as CAMPATH, anti-CD 3 antibodies, or other antibody therapies, cytotoxins, fludarabine (fludarabine), cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation. These drugs inhibit the calcium-dependent phosphatases calcineurin (cyclosporin and FK506), proteasome (bortezomib), or inhibit p70S6 kinase (rapamycin), which is important for growth factor-induced signaling. (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 further embodiments, the cell compositions of the present disclosure are used in conjunction with bone marrow transplantation, with chemotherapeutic agents such asFludarabine, in vitro radiotherapy (XRT), cyclophosphamide or an antibody such as OKT3 or CAMPATH is administered to the patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) T cell ablation therapy (T cell ablation therapy). In one embodiment, in B cell ablative therapies such as agents that react with CD20 such asFollowed by administration of the cell composition of the present disclosure. In one embodiment, the cell composition of the present disclosure is administered after B cell ablation therapy with an agent such as bortezomib. For example, in one embodiment, the subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following transplantation, the subject receives an infusion of the expanded immune cells of the present disclosure. In further embodiments, the expanded cells are administered before or after surgery.

The dosage of the above-described treatments to be administered to a patient will vary with the exact nature of the condition being treated and the recipient of the treatment. The dosage for human administration may be scaled according to art-recognized practices.

The modified B cells are useful for treating or preventing various diseases and disorders. In particular embodiments, B cells modified to express follistatin are used in a method of increasing muscle size or strength in a patient. For example, the present disclosure provides a method of increasing muscle size or strength in a subject, the method comprising administering an effective amount of a B cell that expresses a follistatin polypeptide. The increased muscle size or strength may generally occur throughout the subject, or it may occur in the targeted muscle. The targeted muscle may be damaged, weakened or deficient, as may be the case in a variety of muscle disorders including muscular dystrophy (such as duchenne muscular dystrophy, becker muscular dystrophy, Emery Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, ankylosing spondylitis syndrome, Ulirich syndrome, foishan muscular dystrophy, Walker-Warburg syndrome, myo-eye-encephalopathy, facioscapulohumeral muscular dystrophy), congenital muscular dystrophy, myotonic dystrophy (Steinert's disease), non-dystrophic myotonia, periodic paralysis, spinal muscular atrophy, familial amyotrophic lateral sclerosis, hereditary motor and sensory neuropathy, charc-Marie-Tooth disease, chronic inflammatory neuropathy, distal myopathy, myotube/central myopathy, linear myopathy (neuropathies), nuclear cardiomyopathy, central core disease, desminopathies, inclusion body myositis, mitochondrial myopathy, congenital myasthenia syndrome, post polio muscle dysfunction and Emery (2002) The Lancet,359: 687-; and Khurana et al, (2003) nat. Rev. drug Disc.,2: 379-. For example, in some cases, muscles may be damaged, weakened, or absent due to sarcopenia. In some cases, muscle may be damaged, weakened, or absent due to Spinal Muscular Atrophy (SMA). In some cases, muscles may be damaged, weakened, or absent due to Amyotrophic Lateral Sclerosis (ALS). In some cases, the muscle may be damaged, weakened or deficient due to pompe disease. The method may also increase muscle size or strength in healthy muscle.

In some embodiments, the present disclosure provides methods for treating a disease or disorder in an individual comprising administering to a subject in need thereof a B cell genetically modified to express follistatin (e.g., a follistatin + B cell that expresses an FST344 transcript variant).

In some embodiments, the disclosure provides methods for treating a muscle disorder in an individual, the method comprising administering to a subject having or suspected of having such a muscle disorder a B cell genetically modified to express follistatin (e.g., follistatin + B cells that express an FST344 transcript variant), 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, ankylosing spondylitis, Ulirich syndrome, foie-type muscular dystrophy, Walker-Warburg syndrome, myo-eye-encephalopathy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, myotonic dystrophy (Steinert's disease), non-dystrophic myotonia, periodic paralysis, spinal muscular atrophy, familial amyotrophic lateral sclerosis, hereditary motor and sensory neuropathy, Charcot-Marie-Tooth disease, chronic inflammatory neuropathy, distal myopathy, myotube/central myopathy, mitochondrial myopathy (nemaline myopathy), mini-nuclear disease, central nuclear disease, desminopathy, inclusion body myositis, mitochondrial myopathy, myasthenia syndrome, congenital muscular dystrophy, Post-polio muscle dysfunction and Emery (2002) The Lancet,359: 687-; and Khurana et al, (2003) nat. Rev. drug Disc.,2: 379-.

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

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

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

In some embodiments, the disclosure provides methods for treating a muscle disorder in an individual comprising administering to a subject having or suspected of having such a muscle disorder a B cell genetically modified to express follistatin (e.g., a follistatin + B cell that expresses an FST344 transcript variant), wherein the muscle disorder is selected from muscle atrophy or weakening due to various types of aging, cancer, or chronic disease.

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

In some embodiments, the present disclosure provides methods for treating an individual exhibiting mild, moderate, or severe muscle fragility, muscle hypertrophy, muscle pseudohypertrophy, joint contractures, skeletal deformities, cardiomyopathy, impaired swallowing, impaired bowel and bladder function, muscle ischemia, cognitive impairment, behavioral dysfunction, social impairment, scoliosis, and/or impaired respiratory function comprising administering to the subject a B cell genetically modified to express follistatin (e.g., a follistatin + B cell that expresses an FST344 transcript variant).

In particular embodiments, the disclosure provides methods for treating muscular dystrophy in an individual comprising administering to a subject having or suspected of having becker muscular dystrophy a B cell genetically modified to express follistatin (e.g., follistatin + B cells that express an FST344 transcript variant).

In some embodiments, a single maximum effective dose of follistatin + B cells (e.g., follistatin + B cells that express the FST344 transcript variant) is administered to the subject. In some embodiments, two or more doses of follistatin + B cells (e.g., follistatin + B cells that express the FST344 transcript variant) are administered to the subject, thereby maximizing the amount of follistatin + B cells that are transplanted. In some embodiments, two or more doses of follistatin + B cells (e.g., follistatin + B cells that express the FST344 transcript variant) administered to the subject comprise fewer follistatin + B cells than a single maximally effective dose of follistatin + B cells. In some embodiments, when two or more doses of follistatin + B cells (e.g., follistatin + B cells that express an FST344 transcript variant) are administered to a subject at a dose that is less than the maximum effective single dose of follistatin + B cells, a synergistic increase in follistatin production results. In one embodiment, administration of follistatin + B cells to a subject results in normal levels of follistatin being observed in healthy control subjects. In one embodiment, administration of follistatin + B cells to a subject results in a level of follistatin in the subject that is higher than normal. In one embodiment, administration of follistatin + B cells (e.g., follistatin + B cells that express the FST344 transcript variant) to the subject increases the strength of the subject. In one embodiment, administration of follistatin + B cells (e.g., follistatin + B cells that express the FST344 transcript variant) to the subject increases the strength of the subject compared to normal levels. In one embodiment, administration of follistatin + B cells (e.g., follistatin + B cells that express the FST344 transcript variant) to a subject prevents the subject from losing strength.

Examples

Example 1

Production of follistatin-expressing B cells

Sleeping beauty transposons and transposase constructs for transposition and expression of human Follistatin (FST) were generated. The transposon is assembled to achieve FST gene integration and expression in B cells. We used the EEK promoter (consisting of promoter and enhancer elements from human immunoglobulin genes and other regulatory elements previously described) to achieve high levels of expression in B cells. To test for FST transposition and expression, human B cells were isolated from two separate donors, expanded in B cell culture medium and incubated in a 37 ℃ incubator with 5% CO2, electroporated on day 3 with pKT2/EEK-FST344 plus mRNA encoding SB100x transposase. Cell lysates prepared at day 2, day 5, day 8 and day 11 post-electroporation (i.e., day 5, day 7, day 11 and day 14 of culture) contained significantly increased FST over wild-type non-transfected cells, demonstrating the effectiveness of the SB transposon system in achieving high levels of FST expression in expanded human B cells (fig. 5A). Notably, after a slight decrease from day 5 (probably due to initial episomal FST expression), FST expression persisted from day 7 to day 14, indicating 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). In the examples below, these FST expressing B cells are referred to as FST + B cells.

Example 2

In vivo production of FST

To determine whether B cells engineered according to the present disclosure can promote increased FST production in vivo, we injected wild type mice with FST + B cells produced in example 1.

Specifically, four mice received intravenous (tail vein) injections of vehicle (500 μ Ι Phosphate Buffered Saline (PBS)) or 2 × 10 diluted to 500 μ Ι in vehicle (PBS) on day 0⁶Human FST + B cells. In addition, on day-7, use 3X10⁶A primary autologous peripheral blood cell enriched in CD4+ T cells was intraperitoneally (i.p.) infused into mice to provide support for pKT2/EEK-FST plus mRNA encoding SB100x transposase for transposable B cells. We observed a two-fold increase in human FST in plasma of mice treated with FST + B cells, which provides strong evidence for successful adoptive transfer of human B cells (fig. 1). FST levels peaked at about 28 days post infusion and dropped to near normal levels on day 35. Although not performed in FST-deficient animals, the results from this experiment still provide an example of the levels of human FST that can be achieved after introduction of a highly potent FST + B cell population into wt mice. We found that plasma levels of FST correlate with plasma levels of human IgG; thus, evidence for adoptive transfer and engraftment of FST + B cells was provided (fig. 2A-2D).

To determine if FST + B cells had any effect on the treated mice, we monitored the body weight of control and FST + B cell treated mice 35 days after infusion. As shown in fig. 3, FST + B cells increased by an average of 4.1% (0.9 g) over vehicle control-treated mice. Furthermore, the increase in body weight gain corresponded to the foreleg grip test (16% improvement in FST + B cell treated mice compared to vehicle control) (fig. 4A); four leg grip test (23% improvement in FST + B cell treated mice compared to vehicle control) (fig. 4B); significant force improvement in the hanging test (23% improvement in FST + B cell treated mice compared to vehicle control) (figure 4C).

Thus, these data show that B cells can be used in the methods disclosed herein to express FST and deliver FST to a subject to induce weight gain and strength improvement.

The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the application data sheet, are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary, to employ concepts of the various patents, applications and publications to provide yet further embodiments.

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

Sequence listing

<110> Yimuxofft Corporation (Immunsoft Corporation)

Mohsin Ruien Shu (Scholz, Matthew Rein)

Ehrick J Herbiger (Herbig, Eric J.)

R.Scott.McIvor (R. Scott)

<120> B cells genetically engineered to secrete follistatin and methods of using the same to treat follistatin-related diseases, conditions, disorders, and enhance muscle growth and strength

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

<150> US 62/644,362

<151> 2018-03-16

<150> US 62/644,356

<151> 2018-03-16

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Claims (85)

1. A recombinant B cell comprising a follistatin gene.

2. The B-cell of claim 1, wherein the follistatin gene is operably linked to a promoter.

3. The B-cell of claim 1 or 2, wherein the follistatin gene is a human follistatin gene.

4. The B-cell of any one of claims 1-3, wherein the follistatin gene is a human follistatin FST-344 splice site variant.

5. The B cell of any one of the preceding claims, wherein the B cell is a human B cell.

6. The B cell of any one of the preceding claims, wherein the B cell has been transduced with or transposed with the follistatin gene.

7. The B-cell of any one of claims 1-6, wherein the B-cell comprises the follistatin gene in that the B-cell has been transduced with the follistatin gene using a transposon system.

8. The B cell of claim 7, wherein the transposon system is a sleeping beauty transposon system or a Piggybac transposon system.

9. The B-cell of any one of claims 1-7, wherein the B-cell expresses the follistatin gene as a result of being transduced with a virus that carries the follistatin gene.

10. The B-cell of any one of claims 1-7, wherein the B-cell comprises the follistatin gene in that the B-cell has been transduced with a retrovirus, lentivirus, adenovirus, or adeno-associated virus comprising the follistatin gene.

11. The B-cell of any one of claims 1-7, wherein the B-cell is engineered to contain the follistatin gene using a targeted integration approach.

12. The B-cell of claim 11, wherein the targeted integration utilizes one or more of a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), and/or a CRISPR/Cas system including but not limited to the CRISPR/Cas9 system.

13. The B-cell of any one of claims 1-7, wherein the B-cell is engineered to comprise the 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 vector, non-viral DNA and/or RNA encoding follistatin introduced using chemical or physical means, such as lipofection, polycationic complexation, electroporation, etc.

14. The B-cell of any one of the preceding claims, wherein the follistatin protein is secreted by the recombinant B-cell.

15. A method of delivering follistatin to a subject comprising administering a recombinant B cell comprising a follistatin gene.

16. A method of delivering follistatin to a subject in need thereof, comprising administering the recombinant B cell of any one of claims 1-14.

17. The method of claim 15 or 16, wherein the subject is a mammal.

18. The method of any one of claims 15-17, wherein the subject is a human.

19. The method of any one of claims 15-18, wherein the subject has muscular dystrophy.

20. The method of any one of claims 15-19, wherein the subject has becker muscular dystrophy.

21. The method of any one of claims 15-20, wherein administering the recombinant B cell to the subject achieves treatment of a disease, disorder, or condition in the subject.

22. The method of any one of claims 15-20, wherein administering the recombinant B cell to the subject achieves treatment of muscular dystrophy.

23. The method of any one of claims 15-22, wherein administration of the recombinant B cells to the subject results in an increase in the subject's weight.

24. The method of claim 23, wherein the subject has increased body weight by at least about 4%.

25. The method of claim 24, wherein a significant increase in body weight occurs within 30 days.

26. The method of claim 24, wherein a significant increase in body weight occurs within about 30 days.

27. The method of any one of claims 15-26, wherein administering the recombinant B cells to the subject results in the subject acquiring muscle mass.

28. The method of any one of claims 15-27, wherein administering the recombinant B cells to the subject causes the subject to become more robust.

29. The method of any one of claims 15-28, wherein administration of the recombinant B cell results in an increase in plasma levels of follistatin in the subject.

30. A method of treating, preventing or ameliorating a muscle disorder by administering a recombinant B cell comprising a follistatin gene.

31. A method of treating, preventing or ameliorating muscular dystrophy by administering the recombinant B cell of any one of claims 1-13.

32. The recombinant B cell of any one of claims 1-13, wherein the recombinant B cell is derived from a B cell obtained from the subject or a B cell derived from a cell obtained from the subject.

33. The recombinant B cell of claim 32, wherein the recombinant B cell is derived from a B cell progenitor obtained from the subject.

34. The recombinant B cell of claim 32, wherein the recombinant B cell is derived from a cell obtained from the subject that has dedifferentiated into the B cell or B cell progenitor.

35. The recombinant B cell of any one of claims 1-13 and 32-34, wherein the recombinant B cell is engineered by:

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

(b) transducing said cell with a DNA encoding said follistatin;

(c) expanding selected cells ex vivo; and

(d) (ii) differentiating said expanded cells ex vivo into plasma cells and/or plasmablasts.

36. The recombinant B cell of claim 35, wherein the isolated immune cell from step a is a CD19 positive cell.

37. The recombinant B cell of claim 35 or 36, wherein transduction in step B is performed by electroporation.

38. The recombinant B cell of claim 37, wherein said electroporation utilizes the sleeping beauty transposon system.

39. The recombinant B cell of any one of claims 35-38, wherein the differentiated cell is CD38(+) and CD20 (-).

40. A method comprising administering to a subject the recombinant B cell of any one of claims 35-39.

41. The method of any one of claims 15-31 and 35-40, wherein the method comprises administering two or more consecutive doses of genetically modified B cells to the subject.

42. The method of claim 41, wherein administering comprises two or more doses of genetically modified B cells at sub-optimal single dose concentrations.

43. The method of claim 41, wherein administering 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 of claim 41, wherein the subject is a human.

47. The method of claim 41, wherein the genetically modified B cells are CD20-, CD38-, and CD 138-.

48. The method of 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 CD 138-.

50. The method of claim 41, wherein the administering comprises intravenous injection, intraperitoneal injection, subcutaneous injection, intrathecal injection, intracameral injection, or intramuscular injection.

51. The method of claim 50, wherein said administering comprises intravenous injection.

52. The method of any one of claims 15-31 and 35-51, wherein the genetically modified B cells are engineered on day 2 or day 3 after culture.

53. The method of claim 52, wherein the genetically modified B cell is engineered using a method comprising electroporation.

54. The method of any one of claims 15-31 and 35-53, wherein

(a) Harvesting the genetically modified B cells for administration to a subject on one of days 1 to 12 of in vitro culture,

(b) harvesting the genetically modified B cells for administration to a subject on day 4, day 5, day 6, or day 7, or day 8 of post-engineering culture.

55. The method of any one of claims 15-31 and 35-54, wherein the genetically modified B cells are harvested for administration to a subject on or after day 8 of the initial culture after engineering.

56. The method of claim 55, wherein the genetically modified B cells are harvested for administration to a subject on day 10 or earlier from the start of culture after engineering.

57. The method of any one of claims 15-31 and 35-56, wherein the harvested genetically modified B cells do not produce significant levels of inflammatory cytokines.

58. The method of any one of claims 15-31 and 35-57, wherein the genetically modified B cells are harvested at a culture time point determined that the genetically modified B cells do not produce significant levels of inflammatory cytokines.

59. The method of any one of claims 15-31 and 35-58, wherein the genetically modified B cells are grown in a culture system comprising each of IL-2, IL-4, IL-10, IL-15, IL-31, and a multimerized CD40 ligand throughout the culture period before and after engineering.

60. The method of claim 59, wherein the multimerized CD40 ligand is a HIS-labeled CD40 ligand that is multimerized using an anti-HIS antibody.

61. The method of any one of claims 15-31 and 35-60, further comprising expanding the genetically modified B cells prior to administration to the subject.

62. The method of claim 61, wherein the expanded final population of genetically modified B cells exhibits a high degree of polyclonality.

63. The method of claim 61, wherein any particular B cell clone in the expanded final population of genetically modified B cells comprises less than 0.2% of the total B cell population.

64. The method of claim 61, wherein any particular B cell clone in the expanded final population of genetically modified B cells comprises less than 0.05% of the total B cell population.

65. The method of any one of claims 15-31 and 35-64, wherein the genetically modified B cell comprises a polynucleotide encoding a selectable marker.

66. The method of claim 65, wherein said selectable marker is a human DHFR gene with enhanced resistance to methotrexate.

67. The method of claim 66, wherein the human DHFR gene having increased resistance to methotrexate contains a leucine to tyrosine substitution mutation at amino acid 22 and a phenylalanine to serine substitution mutation at amino acid 31.

68. The method of any one of claims 15-31 and 35-67, comprising treating the genetically modified B cells with methotrexate prior to harvesting for administration.

69. The method of claim 68, wherein the methotrexate treatment is 100nM to 300 nM.

70. The method of claim 69, wherein the methotrexate treatment is 200 nM.

71. The method of any one of claims 15-31 and 35-70, wherein the genetically modified B cells migrate to multiple tissues after administration to the subject.

72. The method of any one of claims 15-31 and 35-71, wherein at least one genetically modified B cell in the population of genetically modified B cells administered to the subject migrates to one or more tissues selected from the group consisting of: bone marrow, intestine, muscle, spleen, kidney, heart, liver, lung, and brain.

73. The method of claim 72, wherein at least one genetically modified B cell in the population of genetically modified B cells administered to the subject migrates to bone marrow, intestine, muscle, spleen, kidney, heart, liver, lung, and brain of the subject.

74. A modified B cell transduced to express a follistatin gene and a DHFR gene.

75. A method of treating a muscle disorder comprising administering to a subject a B cell genetically modified to express follistatin.

76. The method of claim 75, wherein the muscle disorder is selected from the group consisting of muscular dystrophy, an inflammatory muscle disorder, muscle injury or trauma, muscle disuse, and muscle atrophy or weakening.

77. The method of claim 75 or 76, wherein the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, or facioscapulohumeral muscular dystrophy.

78. The method of claim 75 or 76, wherein the inflammatory muscle disorder is inclusion body myositis.

79. The method of claim 75 or 76, wherein the muscle disuse occurs after extended bed rest or limb immobilization.

80. The method of claim 75 or 76, wherein the muscle atrophy or weakening is caused by aging, cancer or chronic disease.

81. The method of claim 75, wherein the muscle disorder is sarcopenia.

82. The method of claim 75, wherein the muscle disorder is Spinal Muscular Atrophy (SMA).

83. The method of claim 75, wherein the muscle disorder is Amyotrophic Lateral Sclerosis (ALS).

84. The method of claim 75, wherein the muscle disorder is Pompe disease.

85. The method of any one of claims 75-84, wherein the follistatin comprises an amino acid sequence shown in any one of SEQ ID NOs 1-4.