B-CELL ENGINEERING
20190352614 ยท 2019-11-21
Inventors
Cpc classification
A61K35/17
HUMAN NECESSITIES
A61K39/46433
HUMAN NECESSITIES
C12N2750/14143
CHEMISTRY; METALLURGY
C12N5/163
CHEMISTRY; METALLURGY
A61K48/00
HUMAN NECESSITIES
A61K35/28
HUMAN NECESSITIES
A61P7/02
HUMAN NECESSITIES
International classification
A61K35/28
HUMAN NECESSITIES
C12N15/90
CHEMISTRY; METALLURGY
Abstract
Described herein are constructs used for B-cell genomic engineering and for expression of a transgene and/or for modulation of B cell function.
Claims
1. A genetically modified B cell comprising one or more modifications comprising: (a) one or more transgenes, and/or (b) insertions and/or deletions which modify (i) B cell receptor genes, and/or (ii) cellular interactions in Germinal Centers, and/or (c) modifications that inhibit suppression of any B cell function associated with pathogen infection or cancer regulation.
2. The genetically modified B cell of claim 1, wherein one or more of the transgenes are integrated into an endogenous locus of the B cell.
3. The genetically modified B cell of claim 1, wherein the transgene encodes a protein lacking or deficient in a subject with a hemophilia, a lysosomal storage disease, a therapeutic antibody and/or a peptide that facilitates crossing the blood-brain barrier when fused to a therapeutic protein.
4. The genetically modified B cell of claim 3, wherein the therapeutic antibody is specific for a B cell that generates inhibitory antibodies to a protein provided by enzyme replacement therapy (ERT) or acts in an autoimmune disease.
5. The genetically modified B cell of claim 3, wherein the therapeutic antibody is specific for a regulatory B cell (Breg) capable of attenuating an anti-tumor response.
6. The genetically modified B cell of claim 4, wherein the protein provided by ERT is a clotting factor.
7. The genetically modified B cell of claim 6, wherein the clotting factor is Factor IX (F9).
8. The genetically modified B cell of claim 1, wherein the transgene further comprises a promoter that drives expression of the transgene.
9. The genetically modified B cell of claim 8, wherein the promoter is a lineage-specific B cell promoter.
10. The genetically modified B cell of claim 1, wherein a transgene is expressed in the cell.
11. The genetically modified B cell of claim 10, wherein the transgene is integrated into a safe harbor locus selected from the group consisting of AAVS1, TCRA, CCR5 or albumin.
12. A genetically modified B cell of claim 1 descended from a genetically modified hematopoietic stem cell.
13. A method of producing a protein in a subject in need thereof, the method comprising administering a population of B cells according to claim 1 to the subject.
14. The method of claim 13, wherein the protein modulates an antibody response in the subject.
15. A method of producing a protein in a subject, the method comprising: introducing into the subject the B cell of claim 1 thereof under conditions such that the B cell produces the protein in the subject.
16. The method of claim 15, wherein the protein is a protein lacking or deficient in a disease or disorder such as a hemophilia or lysosomal storage disease or autoimmune disease or an antibody specific for a B cell producing antibodies to a therapeutic protein supplied in ERT.
17. The method of claim 16, wherein the therapeutic protein supplied in ERT is a clotting factor such as Factor IX (F9) and the antibody is specific for B cells producing anti-clotting factor (anti-F9) antibodies.
18. A kit comprising one or more of the B cells of claim 1.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0052] Disclosed herein are methods and compositions for genetic engineering of a B cell, including knocking out endogenous genes and inserting (stably or episomally) expression cassettes for expression of a transgene. The methods can be carried out in vitro, ex vivo or in vivo and can be used to express any transgene(s) for the treatment and/or prevention of any disease or disorder which can be ameliorated by the provision of one or more of the transgenes.
General
[0053] Practice of the methods, as well as preparation and use of the compositions disclosed herein employ, unless otherwise indicated, conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, computational chemistry, cell culture, recombinant DNA and related fields as are within the skill of the art. These techniques are fully explained in the literature. See, for example, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (P. M. Wassarman and A. P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (P. B. Becker, ed.) Humana Press, Totowa, 1999.
Definitions
[0054] The terms nucleic acid, polynucleotide, and oligonucleotide are used interchangeably and refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form. For the purposes of the present disclosure, these terms are not to be construed as limiting with respect to the length of a polymer. The terms can encompass known analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties (e.g., phosphorothioate backbones). In general, an analogue of a particular nucleotide has the same base-pairing specificity; i.e., an analogue of A will base-pair with T.
[0055] The terms polypeptide, peptide and protein are used interchangeably to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of a corresponding naturally-occurring amino acids.
[0056] Recombination refers to a process of exchange of genetic information between two polynucleotides, including but not limited to, capture by non-homologous end joining (NHEJ) and homologous recombination. For the purposes of this disclosure, homologous recombination (HR) refers to the specialized form of such exchange that takes place, for example, during repair of double-strand breaks in cells via homology-directed repair mechanisms.
[0057] In certain methods of the disclosure, one or more targeted nucleases as described herein create a double-stranded break (DSB) in the target sequence (e.g., cellular chromatin) at a predetermined site (e.g., albumin gene). The DSB mediates integration of a construct as described herein. Optionally, the construct has homology to the nucleotide sequence in the region of the break. The expression construct may be physically integrated or, alternatively, the expression cassette is used as a template for repair of the break via homologous recombination, resulting in the introduction of all or part of the nucleotide sequence as in the expression cassette into the cellular chromatin. Thus, a first sequence in cellular chromatin can be altered and, in certain embodiments, can be converted into a sequence present in an expression cassette. Thus, the use of the terms replace or replacement can be understood to represent replacement of one nucleotide sequence by another, (i.e., replacement of a sequence in the informational sense), and does not necessarily require physical or chemical replacement of one polynucleotide by another.
[0058] In any of the methods described herein, the exogenous nucleotide sequence (the expression construct or expression cassette or vector) can contain sequences that are homologous, but not identical, to genomic sequences in the region of interest, thereby stimulating homologous recombination to insert a non-identical sequence in the region of interest. Thus, in certain embodiments, portions of the expression cassette sequence that are homologous to sequences in the region of interest exhibit between about 80 to 99% (or any integer therebetween) sequence identity to the genomic sequence that is replaced. In other embodiments, the homology between the expression cassette and genomic sequence is higher than 99%, for example if only 1 nucleotide differs as between the homology regions of the expression cassette and genomic sequences of over 100 contiguous base pairs. In certain cases, a non-homologous portion of the expression cassette can contain sequences not present in the region of interest, such that new sequences are introduced into the region of interest. In these instances, the non-homologous sequence is generally flanked by sequences of 50-1,000 base pairs (or any integral value therebetween) or any number of base pairs greater than 1,000, that are homologous or identical to sequences in the region of interest.
[0059] The term sequence refers to a nucleotide sequence of any length, which can be DNA or RNA; can be linear, circular or branched and can be either single-stranded or double stranded. The term transgene refers to a nucleotide sequence that is inserted into a genome. A transgene can be of any length, for example between 2 and 100,000,000 nucleotides in length (or any integer value therebetween or thereabove), preferably between about 100 and 100,000 nucleotides in length (or any integer therebetween), more preferably between about 2000 and 20,000 nucleotides in length (or any value therebetween) and even more preferable, between about 5 and 15 kb (or any value therebetween).
[0060] A chromosome, is a chromatin complex comprising all or a portion of the genome of a cell. The genome of a cell is often characterized by its karyotype, which is the collection of all the chromosomes that comprise the genome of the cell. The genome of a cell can comprise one or more chromosomes.
[0061] An episome is a replicating nucleic acid, nucleoprotein complex or other structure comprising a nucleic acid that is not part of the chromosomal karyotype of a cell. Examples of episomes include plasmids and certain viral genomes. The liver specific constructs described herein may be episomally maintained or, alternatively, may be stably integrated into the cell.
[0062] An exogenous molecule is a molecule that is not normally present in a cell, but can be introduced into a cell by one or more genetic, biochemical or other methods. Normal presence in the cell is determined with respect to the particular developmental stage and environmental conditions of the cell. Thus, for example, a molecule that is present only during embryonic development of muscle is an exogenous molecule with respect to an adult muscle cell. Similarly, a molecule induced by heat shock is an exogenous molecule with respect to a non-heat-shocked cell. An exogenous molecule can comprise, for example, a functioning version of a malfunctioning endogenous molecule or a malfunctioning version of a normally-functioning endogenous molecule.
[0063] An exogenous molecule can be, among other things, a small molecule, such as is generated by a combinatorial chemistry process, or a macromolecule such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules. Nucleic acids include DNA and RNA, can be single- or double-stranded; can be linear, branched or circular; and can be of any length. Nucleic acids include those capable of forming duplexes, as well as triplex-forming nucleic acids. See, for example, U.S. Pat. Nos. 5,176,996 and 5,422,251. Proteins include, but are not limited to, DNA-binding proteins, transcription factors, chromatin remodeling factors, methylated DNA binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, ligases, deubiquitinases, integrases, recombinases, ligases, topoisomerases, gyrases and helicases.
[0064] An exogenous molecule can be the same type of molecule as an endogenous molecule, e.g., an exogenous protein or nucleic acid. For example, an exogenous nucleic acid can comprise an infecting viral genome, a plasmid or episome introduced into a cell, or a chromosome that is not normally present in the cell. Methods for the introduction of exogenous molecules into cells are known to those of skill in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer. An exogenous molecule can also be the same type of molecule as an endogenous molecule but derived from a different species than the cell is derived from. For example, a human nucleic acid sequence may be introduced into a cell line originally derived from a mouse or hamster.
[0065] By contrast, an endogenous molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions. For example, an endogenous nucleic acid can comprise a chromosome, the genome of a mitochondrion, chloroplast or other organelle, or a naturally-occurring episomal nucleic acid. Additional endogenous molecules can include proteins, for example, transcription factors and enzymes.
[0066] As used herein, the term product of an exogenous nucleic acid includes both polynucleotide and polypeptide products, for example, transcription products (polynucleotides such as RNA) and translation products (polypeptides).
[0067] A fusion molecule is a molecule in which two or more subunit molecules are linked, preferably covalently. The subunit molecules can be the same chemical type of molecule, or can be different chemical types of molecules. Examples of fusion molecules include, but are not limited to, fusion proteins (for example, a fusion between a protein DNA-binding domain and a cleavage domain), fusions between a polynucleotide DNA-binding domain (e.g., sgRNA) operatively associated with a cleavage domain, and fusion nucleic acids (for example, a nucleic acid encoding the fusion protein).
[0068] Expression of a fusion protein in a cell can result from delivery of the fusion protein to the cell or by delivery of a polynucleotide encoding the fusion protein to a cell, wherein the polynucleotide is transcribed, and the transcript is translated, to generate the fusion protein. Trans-splicing, polypeptide cleavage and polypeptide ligation can also be involved in expression of a protein in a cell. Methods for polynucleotide and polypeptide delivery to cells are presented elsewhere in this disclosure.
[0069] A gene, for the purposes of the present disclosure, includes a DNA region encoding a gene product (see infra), as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions.
[0070] Gene expression refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.
[0071] Modulation of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression. Genome editing (e.g., cleavage, alteration, inactivation, random mutation) can be used to modulate expression. Gene inactivation refers to any reduction in gene expression as compared to a cell that does not include a ZFP, TALE or CRISPR/Cas system as described herein. Thus, gene inactivation may be partial or complete. A genetically modified cell includes cells with any change to the genetic material in the cell, including but not limited to episomal and/or genomic modifications. Non-limiting examples of genetic modifications includes insertions and/or deletions (for example episomal and/or targeted integration of one or more transgenes, RNAs or non-coding sequences) and/or mutations (for example point mutations, substitutions, etc.) that alter protein expression within the cell).
[0072] A region of interest is any region of cellular chromatin, such as, for example, a gene or a non-coding sequence within or adjacent to a gene, in which it is desirable to bind an exogenous molecule. Binding can be for the purposes of targeted DNA cleavage and/or targeted recombination. A region of interest can be present in a chromosome, an episome, an organellar genome (e.g., mitochondrial, chloroplast), or an infecting viral genome, for example. A region of interest can be within the coding region of a gene, within transcribed non-coding regions such as, for example, leader sequences, trailer sequences or introns, or within non-transcribed regions, either upstream or downstream of the coding region. A region of interest can be as small as a single nucleotide pair or up to 2,000 nucleotide pairs in length, or any integral value of nucleotide pairs.
[0073] Eukaryotic cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells (e.g., B-cells), including stem cells (pluripotent and multipotent).
[0074] The terms operative linkage and operatively linked (or operably linked) are used interchangeably with reference to a juxtaposition of two or more components (such as sequence elements), in which the components are arranged such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. By way of illustration, a transcriptional regulatory sequence, such as a promoter, is operatively linked to a coding sequence if the transcriptional regulatory sequence controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. A transcriptional regulatory sequence is generally operatively linked in cis with a coding sequence, but need not be directly adjacent to it. For example, an enhancer is a transcriptional regulatory sequence that is operatively linked to a coding sequence, even though they are not contiguous.
[0075] A functional fragment of a protein, polypeptide or nucleic acid is a protein, polypeptide or nucleic acid whose sequence is not identical to the full-length protein, polypeptide or nucleic acid, yet retains the same function as the full-length protein, polypeptide or nucleic acid. A functional fragment can possess more, fewer, or the same number of residues as the corresponding native molecule, and/or can contain one or more amino acid or nucleotide substitutions. Methods for determining the function of a nucleic acid (e.g., coding function, ability to hybridize to another nucleic acid) are well-known in the art. Similarly, methods for determining protein function are well-known. For example, the B-domain deleted human Factor VIII is a functional fragment of the full-length Factor VIII protein.
[0076] A polynucleotide vector or construct is capable of transferring gene sequences to target cells. Typically, vector construct, expression vector, expression construct, expression cassette, and gene transfer vector, mean any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells. Thus, the term includes cloning, and expression vehicles, as well as integrating vectors.
[0077] The terms subject and patient are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, dogs, cats, rats, mice, and other animals. Accordingly, the term subject or patient as used herein means any mammalian patient or subject to which the expression cassettes of the invention can be administered. Subjects of the present invention include those with a disorder.
B Cell Expression Constructs
[0078] Described herein are expression cassettes (constructs) for use in directing expression of a transgene in a B cell (including plasmablasts and plasma cells), including in vivo following administration of the expression cassette(s) to the subject (e.g., intravenous delivery). The expression construct may be maintained episomally and drive expression of the transgene extrachromosomally or, alternatively, the expression construct may be integrated into the genome of a B cell, for example by nuclease-mediated targeted integration.
[0079] Any suitable promoter sequence can be used in the expression cassettes of the invention. In certain embodiments, the promoter is a constitutive promoter. In other embodiments, the promoter is inducible and/or is a B cell specific promoter. Promoterless constructs in which the transgene is driven by an endogenous B cell promoter are also contemplated for genetic modification of cells as described herein.
[0080] As will be apparent, any transgene can be used in the constructs described herein. Furthermore, the individual expression construct components (promoter, enhancer, insulator, intron, transgene, etc.) of the constructs described herein may be present or not, and may mixed and matched in any combination.
[0081] The constructs described herein may be contained within any viral or non-viral vector. The constructs may be maintained episomally or may be integrated into the genome of the cell (e.g., via nuclease-mediated targeted integration).
[0082] Non-viral vectors include DNA or RNA plasmids, DNA MCs, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome, lipid nanoparticle, nanoparticle or poloxamer. Viral vectors that may be used to carry the expression cassettes described herein include, but are not limited to, retroviral, lentivirus, adenoviral, adeno-associated viral vectors, vaccinia and herpes simplex virus vectors. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, and as described herein may be facilitated by nuclease-mediated integration.
[0083] In certain preferred embodiments, the constructs are included in an adeno-associated virus (AAV) vector or vector system that may be maintained episomally or integrated into the genome of a B cell (e.g., via nuclease-mediated targeted integration). Construction of recombinant AAV vectors is in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989).
[0084] Thus, in certain embodiments, the expression construct is carried on an AAV construct and further comprises 5 and 3 ITRs flanking the expression constructs elements (e.g., enhancer, promoter, optional intron, transgene, etc.) as described herein. Optionally, spacer molecules are also included between one or more of the components of the expression construct, for example, between the 5 ITR and the enhancer and/or between the polyadenylation signal and the 3 ITR. The spacers may function as homology arms to facilitate recombination into a safe-harbor locus (e.g. albumin). In certain embodiments, the construct is a construct as shown in
[0085] In certain embodiments, the AAV vectors as described herein can be derived from any AAV. In certain embodiments, the AAV vector is derived from the defective and nonpathogenic parvovirus adeno-associated type 2 virus. All such vectors are derived from a plasmid that retains only the AAV 145 bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genomes of the transduced cell are key features for this vector system. (Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther. 9:748-55 (1996)). Other AAV serotypes, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and AAVrh.10 and any novel AAV serotype can also be used in accordance with the present invention. Especially preferred are AAV6 serotypes. In some embodiments, chimeric AAV is used where the viral origins of the ITR sequences of the viral nucleic acid are heterologous to the viral origin of the capsid sequences. Non-limiting examples include chimeric virus with ITR derived from AAV2 and capsids derived from AAV5, AAV6, AAV8 or AAV9 (i.e. AAV2/5, AAV2/6, AAV2/8 and AAV2/9, respectively).
[0086] Retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700).
[0087] The constructs described herein may also be incorporated into an adenoviral vector system. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and high levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system.
[0088] pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn et al., Nat. Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138 (1997)). PA317/pLASN was the first therapeutic vector used in a gene therapy trial. (Blaese et al., Science 270:475-480 (1995)). Transduction efficiencies of 50% or greater have been observed for MFG-S packaged vectors. (Ellem et al., Immunol Immunother. 44(1):10-20 (1997); Dranoff et al., Hum. Gene Ther. 1:111-2 (1997).
[0089] Replication-deficient recombinant adenoviral vectors (Ad) can also be used with the polynucleotides described herein. Most adenovirus vectors are engineered such that a transgene replaces the Ad E1a, E1b, and/or E3 genes; subsequently the replication defective vector is propagated in human 293 cells that supply deleted gene function in trans. Ad vectors can transduce multiple types of tissues in vivo, including nondividing, differentiated cells such as those found in liver, kidney and muscle. Conventional Ad vectors have a large carrying capacity. An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the use of adenovirus vectors for gene transfer in clinical trials include Rosenecker et al., Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:7 1083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarez et al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther. 5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).
[0090] Packaging cells are used to form virus particles that are capable of infecting a host cell. Such cells include HEK293 and Sf9 cells, which can be used to package AAV and adenovirus, and ?2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by a producer cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host (if applicable), other viral sequences being replaced by an expression cassette encoding the protein to be expressed. The missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess inverted terminal repeat (ITR) sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. In some embodiments, AAV is produced using a baculovirus expression system (see e.g. U.S. Pat. Nos. 6,723,551 and 7,271,002).
[0091] Purification of AAV particles from a 293 or baculovirus system typically involves growth of the cells which produce the virus, followed by collection of the viral particles from the cell supernatant or lysing the cells and collecting the virus from the crude lysate. AAV is then purified by methods known in the art including ion exchange chromatography (e.g. see U.S. Pat. Nos. 7,419,817 and 6,989,264), ion exchange chromatography and CsCl density centrifugation (e.g. PCT publication WO2011094198A10), immunoaffinity chromatography (e.g. WO2016128408) or purification using AVB Sepharose (e.g. GE Healthcare Life Sciences).
[0092] In many gene therapy applications, it is desirable that the gene therapy vector be delivered with a high degree of specificity to a particular tissue type. Accordingly, a viral vector can be modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the outer surface of the virus. The ligand is chosen to have affinity for a receptor known to be present on the cell type of interest. For example, Han et al., Proc. Natl. Acad. Sci. USA 92:9747-9751 (1995), reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor. This principle can be extended to other virus-target cell pairs, in which the target cell expresses a receptor and the virus expresses a fusion protein comprising a ligand for the cell-surface receptor. For example, filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor. Although the above description applies primarily to viral vectors, the same principles can be applied to nonviral vectors. Such vectors can be engineered to contain specific uptake sequences which favor uptake by specific target cells.
[0093] The polynucleotides described herein may include one or more non-natural bases and/or backbones. In particular, an expression cassette as described herein may include methylated cytosines to achieve a state of transcriptional quiescence in a region of interest.
[0094] Furthermore, the expression constructs as described herein may also include additional transcriptional or translational regulatory or other sequences, for example, Kozak sequences, additional promoters, enhancers, insulators, introns, internal ribosome entry sites, sequences encoding 2A peptides, furin cleavage sites and/or polyadenylation signals. Further, the control elements of the genes of interest can be operably linked to reporter genes to create chimeric genes (e.g., reporter expression cassettes).
Modifications
[0095] Described herein are genetically modified B cells comprising one or more of the following modifications: (a) the provision in the cell of one or more transgenes (episomal and/or integrated in any combinations); (b) insertions and/or deletions in one or more genes which modify (i) B cell receptor genes, and/or (ii) cellular interactions in Germinal Centers; and/or (c) modifications (mutations) that inhibit suppression of any B cell function associated with pathogen infection or cancer regulation. Genetically modified B cells as described herein may also be descended from HSCs comprising one or more of these genetic modifications.
[0096] In certain embodiments, the constructs described herein can be used for B cell expression of any transgene(s). One or more transgenes may be expressed episomally in the modified B cells and/or following nuclease-mediated targeted integration of one or more of the transgenes. Exemplary transgenes (also referred to as genes of interest and/or exogenous sequences) include, but are not limited to any polypeptide coding sequence (e.g., cDNAs), promoter sequences, enhancer sequences, epitope tags, marker genes, cleavage enzyme recognition sites and/or various types of expression constructs. Marker genes include, but are not limited to, sequences encoding proteins that mediate antibiotic resistance (e.g., ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance), sequences encoding colored or fluorescent or luminescent proteins (e.g., green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, luciferase), and proteins which mediate enhanced cell growth and/or gene amplification (e.g., dihydrofolate reductase). Epitope tags include, for example, one or more copies of FLAG, His, myc, Tap, HA or any detectable amino acid sequence.
[0097] In a preferred embodiment, the transgene comprises a polynucleotide encoding any polypeptide of which expression in the cell is desired, including, but not limited to antibodies, antigens, enzymes, receptors (cell surface or nuclear), hormones, lymphokines, cytokines, reporter polypeptides, growth factors, and functional fragments of any of the above. The coding sequences may be, for example, cDNAs.
[0098] In certain embodiments, the transgene(s) encode(s) functional versions of proteins lacking of deficient in any genetic disease, including but not limited to, lysosomal storage disorders (e.g., Gaucher, Fabry, Hunter, Hurler, Neimann-Pick, etc.), metabolic disorders, and/or blood disorders such as hemophilias and hemoglobinopathies, etc. See, e.g., U.S. Publication No. 20140017212 and 20140093913; U.S. Pat. Nos. 9,255,250 and 9,175,280.
[0099] For example, the transgene may comprise a sequence encoding a polypeptide that is lacking or non-functional in the subject having a genetic disease, including but not limited to any of the following genetic diseases: achondroplasia, achromatopsia, acid maltase deficiency, adenosine deaminase deficiency (OMIM No. 102700), adrenoleukodystrophy, aicardi syndrome, alpha-1 antitrypsin deficiency, alpha-thalassemia, androgen insensitivity syndrome, apert syndrome, arrhythmogenic right ventricular, dysplasia, ataxia telangictasia, barth syndrome, beta-thalassemia, blue rubber bleb nevus syndrome, canavan disease, chronic granulomatous diseases (CGD), cri du chat syndrome, cystic fibrosis, dercum's disease, ectodermal dysplasia, fanconi anemia, fibrodysplasiaossificans progressive, fragile X syndrome, galactosemis, Gaucher's disease, generalized gangliosidoses (e.g., GM1), hemochromatosis, the hemoglobin C mutation in the 6.sup.th codon of beta-globin (HbC), hemophilia, Huntington's disease, Hurler Syndrome, hypophosphatasia, Klinefleter syndrome, Krabbes Disease, Langer-Giedion Syndrome, leukocyte adhesion deficiency (LAD, OMIM No. 116920), leukodystrophy, long QT syndrome, Marfan syndrome, Moebius syndrome, mucopolysaccharidosis (MPS), nail patella syndrome, nephrogenic diabetes insipdius, neurofibromatosis, Neimann-Pick disease, osteogenesisimperfecta, porphyria, Prader-Willi syndrome, progeria, Proteus syndrome, retinoblastoma, Rett syndrome, Rubinstein-Taybi syndrome, Sanfilippo syndrome, severe combined immunodeficiency (SCID), Shwachman syndrome, sickle cell disease (sickle cell anemia), Smith-Magenis syndrome, Stickler syndrome, Tay-Sachs disease, Thrombocytopenia Absent Radius (TAR) syndrome, Treacher Collins syndrome, trisomy, tuberous sclerosis, Turner's syndrome, urea cycle disorder, von Hippel-Landau disease, Waardenburg syndrome, Williams syndrome, Wilson's disease, Wiskott-Aldrich syndrome, X-linked lymphoproliferative syndrome (XLP, OMIM No. 308240), acquired immunodeficiencies, lysosomal storage diseases (e.g., Gaucher's disease, GM1, Fabry disease and Tay-Sachs disease), mucopolysaccahidosis (e.g. Hunter disease, Hurler disease), hemoglobinopathies (e.g., sickle cell diseases, HbC, ?-thalassemia, ?-thalassemia) and hemophilias.
[0100] Non-limiting examples of proteins (including functional fragments thereof such as truncated versions) that may be expressed as described herein include fibrinogen, prothrombin, tissue factor, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII (Hageman factor), Factor XIII (fibrin-stabilizing factor), von Willebrand factor, prekallikrein, high molecular weight kininogen (Fitzgerald factor), fibronectin, antithrombin III, heparin cofactor II, protein C, protein S, protein Z, protein Z-related protease inhibitor, plasminogen, alpha 2-antiplasmin, tissue plasminogen activator, urokinase, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, glucocerebrosidase (GBA), ?-galactosidase A (GLA), iduronate sulfatase (IDS), iduronidase (IDUA), acid sphingomyelinase (SMPD1), MMAA, MMAB, MMACHC, MMADHC (C2orf25), MTRR, LMBRD1, MTR, propionyl-CoA carboxylase (PCC) (PCCA and/or PCCB subunits), a glucose-6-phosphate transporter (G6PT) protein or glucose-6-phosphatase (G6Pase), an LDL receptor (LDLR), ApoB, LDLRAP-1, a PCSK9, a mitochondrial protein such as NAGS (N-acetylglutamate synthetase), CPS1 (carbamoyl phosphate synthetase I), and OTC (ornithine transcarbamylase), ASS (argininosuccinic acid synthetase), ASL (argininosuccinase acid lyase) and/or ARG1 (arginase), and/or a solute carrier family 25 (SLC25A13, an aspartate/glutamate carrier) protein, a UGT1A1 or UDP glucuronsyltransferase polypeptide A1, a fumarylacetoacetate hydrolyase (FAH), an alanine-glyoxylate aminotransferase (AGXT) protein, a glyoxylate reductase/hydroxypyruvate reductase (GRHPR) protein, a transthyretin gene (TTR) protein, an ATP7B protein, a phenylalanine hydroxylase (PAH) protein, a lipoprotein lyase (LPL) protein, an engineered nuclease, an engineered transcription factor and/or a therapeutic single chain antibody.
[0101] In other embodiments, the engineered B-cells described herein include one or more transgenes encoding one or more antibodies that are engineered molecules designed to target immune cells via specific molecular targets expressed on cell surfaces. In some embodiments, the engineered B-cells express antibodies designed to target endogenous B cells. These antibodies may induce antibody meditated killing (e.g., through ADCC or complement mediated killing) of B cells or other immune cells involved in attenuating an immune response.
[0102] B cells as described herein can be genetically modified to produce one or more antibodies that are specific for B cells producing undesirable antibodies. Non-limiting examples of B cells producing undesirable antibodies include B cells producing antibodies against proteins administered in ERT (clotting factors such as F8, F9, etc. in hemophilia patients, and/or proteins lacking or deficient in lysosomal storage disorders). The antibody-encoding constructs are introduced into the B-cell precursor or B-cell ex vivo, such that when the cell is re-introduced into the patient the antibody producing B-cells specifically target cells (B cells) producing the protein (e.g. antibody) bound by the engineered antibody. In certain embodiments, the engineered antibody is specific for antibodies directed against a therapeutic protein supplied exogenously (via ERT and/or gene therapy) such that the antibodies against the therapeutic proteins are neutralized. Thus, the compositions and methods described herein include engineered B-cells that produce antibodies that specifically target antibodies (e.g., anti-F9 antibodies) produced by in the patient. The engineered B-cells of these compositions and methods may be administered to the subject as mature B-cells, or as precursor cells (such as HSCs or lymphoid progenitor cells) that differentiate in the subject after administration or, alternatively, may be genetically modified in vivo. In still further embodiments, the proteins produced from the transgenes (for example anti-ERT antibodies) of the genetically modified B-cells are isolated an administered to the subject in need thereof, for example a patient in need of antibodies to the anti-ERT antibodies their body has generated.
[0103] In still further embodiments, the transgene may be an antibody specific for a B cell that is sensitive to a protein involved in an autoimmune disease. The term autoimmune disease refers to any disease or disorder in which the subject mounts a destructive immune response against its own tissues. Autoimmune disorders can affect almost every organ system in the subject (e.g., human), including, but not limited to, diseases of the nervous, gastrointestinal, and endocrine systems, as well as skin and other connective tissues, eyes, blood and blood vessels. Examples of autoimmune diseases include, but are not limited to Hashimoto's thyroiditis, Systemic lupus erythematosus, Sjogren's syndrome, Graves' disease, Scleroderma, Rheumatoid arthritis, Multiple sclerosis, Myasthenia gravis and Diabetes. Thus, the B cells as described herein can comprise a molecule (e.g., engineered antibody) directed to a B cell population in a subject that is sensitive to (and produces antibodies against) an autoantigen involved in an autoimmune disease, including but not limited to myelin basic protein (MBP), insulin, ANA, joint or muscle proteins, thyroid proteins and the like.
[0104] In certain embodiments, the transgene can comprise a marker gene (described above), allowing selection of cells that have undergone targeted integration, and a linked sequence encoding an additional functionality. Non-limiting examples of marker genes include GFP, drug selection marker(s) and the like.
[0105] The constructs described herein may also be used for delivery of non-coding transgenes. Sequences encoding antisense RNAs, RNAi, shRNAs and micro RNAs (miRNAs) may also be used for targeted insertions.
[0106] In certain embodiments, the transgene includes sequences (e.g., coding sequences, also referred to as transgenes) greater than 1 kb in length, for example between 2 and 200 kb, between 2 and 10 kb (or any value therebetween). The transgene may also include one or more nuclease target sites. The transgene may also comprise one or more homology arms. The homology arms comprise sequences with a high degree of homology to those flanking a nuclease cleavage target site. A homology arm can comprise 50, 100, 200, 500, 1000, 2000 or more nucleotides or any value therebetween.
[0107] When integrated (e.g., via nuclease-mediate integration), the transgene may be inserted into an endogenous gene such that all, some or none of the endogenous gene is expressed.
Nucleases
[0108] As noted above, the expression cassettes may be maintained episomally or may be integrated into the genome of the cell. Integration may be random. In certain embodiments, integration of the transgene construct(s) is targeted to a specified gene following cleavage of the target gene by one or more nucleases (e.g., zinc finger nucleases (ZFNs), TALENs, TtAgo, CRISPR/Cas nuclease systems, and homing endonucleases) and the construct integrated by either homology directed repair (HDR) or by end capture during non-homologous end joining (NHEJ) driven processes. See, e.g., U.S. Pat. Nos. 9,394,545; 9,150,847; 9,206,404; 9,045,763; 9,005,973; 8,956,828; 8,936,936; 8,945,868; 8,871,905; 8,586,526; 8,563,314; 8,329,986; 8,399,218; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,067,317; 7,262,054; 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; U.S. Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060063231; 20080159996; 20100218264; 20120017290; 20110265198; 20130137104; 20130122591; 20130177983 and 20130177960 and 20150056705, the disclosures of which are incorporated by reference in their entireties for all purposes.
[0109] Any nuclease can be used for targeted integration of the transgene expression construct.
[0110] In certain embodiments, the nuclease comprises a zinc finger nuclease (ZFN), which comprises a zinc finger DNA-binding domain and a cleavage (nuclease) domain. See, e.g., U.S. Pat. Nos. 9,255,250; 9,200,266; 9,045,763; 9,005,973; 9,150,847; 8,956,828; 8,945,868; 8,703,489; 8,586,526; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,067,317; 7,262,054; 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861.
[0111] In other embodiments, the nuclease comprises a TALEN, which comprises a TAL-effector DNA binding domain and a cleavage (nuclease) domain. See, e.g., U.S. Pat. No. 8,586,526 and U.S. Publication No. 20130196373.
[0112] In still further embodiments, the nuclease comprises a CRISPR/Cas nuclease system, which includes a single guide RNA for recognition of the target site and one or more cleavage domains. See, e.g., U.S. Patent Publication No. 20150056705. In some embodiments, the CRISPR-Cpfl system is used (see Fagerlund et al, (2015) Genom Bio 16:251). It is understood that the term CRISPR/Cas system refers to both CRISPR/Cas and CRISPR/Cfpl systems.
[0113] The cleavage domains of the nucleases may be wild-type or mutated, including non-naturally occurring (engineered) cleavage domains that form obligate heterodimers. See, e.g., U.S. Pat. Nos. 8,623,618; 7,888,121; 7,914,796; and 8,034,598 and U.S. Publication No. 20110201055.
[0114] The nuclease(s) may make one or more double-stranded and/or single-stranded cuts in the target site. In certain embodiments, the nuclease comprises a catalytically inactive cleavage domain (e.g., FokI and/or Cas protein). See, e.g., U.S. Pat. Nos. 9,200,266; 8,703,489 and Guillinger et al. (2014) Nature Biotech. 32(6):577-582. The catalytically inactive cleavage domain may, in combination with a catalytically active domain act as a nickase to make a single-stranded cut. Therefore, two nickases can be used in combination to make a double-stranded cut in a specific region. Additional nickases are also known in the art, for example, McCaffery et al. (2016) Nucleic Acids Res. 44(2):el11. doi: 10.1093/nar/gkv878. Epub 2015 Oct. 19.
[0115] In certain embodiments, the nuclease cleaves a safe harbor gene (e.g., CCR5, Rosa, albumin, AAVS1, TCRA, TCRB, etc. See, e.g., U.S. Pat. Nos. 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; 8,586,526; U.S. Patent Publications 20030232410; 20050208489; 20050026157; 20060063231; 20080159996; 201000218264; 20120017290; 20110265198; 20130137104; 20130122591; 20130177983 and 20130177960. In preferred embodiments, the nuclease cleaves an endogenous albumin gene such that the expression cassette is integrated into the endogenous albumin locus of a liver cell. Albumin-specific nucleases are described, for example, in U.S. Pat. No. 9,150,847; and U.S. Publication Nos. 20130177983 and 20150056705.
Delivery
[0116] The constructs described herein (and/or nucleases) may be delivered in vivo by any suitable means into any cell type, preferably to the spleen or secondary lymph nodes. Similarly, when used in combination with nucleases for targeted integration, the nucleases may be delivered in polynucleotide and/or protein form, for example using non-viral vector(s), viral vectors(s) and/or in RNA form, e.g., as mRNA.
[0117] Methods of non-viral delivery of nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, lipid nanoparticles, immunoliposomes, other nanoparticle, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids. Additional exemplary nucleic acid delivery systems include those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) and Copernicus Therapeutics Inc., (see for example U.S. Pat. No. 6,008,336).
[0118] In preferred embodiments, the expression constructs are AAV vectors. The optional nucleases may be administered in mRNA form or using one or more viral vectors (AAV, Ad, etc.). Administration can be by any means in which the polynucleotides are delivered to the desired target cells. Both in vivo and ex vivo methods are contemplated. Intravenous injection in a peripheral blood vessel is a preferred method of administration. Other in vivo administration modes include, for example, direct injection into tissues comprising B cells including lymph nodes, bone marrow, plasma, lymphatic system and the spleen.
[0119] In systems involving delivery of more than one polynucleotides (e.g., construct as described herein and nuclease in polynucleotide form), the two or more polynucleotide(s) are delivered using one or more of the same and/or different vectors. For example, the nuclease in polynucleotide form may be delivered in mRNA form and the B-cell-specific constructs as described herein may be delivered via other modalities such as viral vectors (e.g., AAV), minicircle DNA, plasmid DNA, linear DNA, liposomes, lipid nanoparticles, nanoparticles and the like.
[0120] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions available, as described below (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989).
[0121] The effective amount of expression cassette (and optional nuclease(s), and/or modified cells) to be administered will vary from patient to patient. Accordingly, effective amounts are best determined by the physician administering the compositions (e.g., cells) and appropriate dosages can be determined readily by one of ordinary skill in the art. Analysis of the serum, plasma or other tissue levels of the therapeutic polypeptide and comparison to the initial level prior to administration can determine whether the amount being administered is too low, within the right range or too high. Suitable regimes for initial and subsequent administrations are also variable, but are typified by an initial administration followed by subsequent administrations if necessary. Subsequent administrations may be administered at variable intervals, ranging from daily to annually to every several years. One of skill in the art will appreciate that appropriate immunosuppressive techniques may be recommended to avoid inhibition or blockage of transduction by immunosuppression of the delivery vectors, see e.g., Vilquin et al., (1995) Human Gene Ther., 6:1391-1401.
[0122] Formulations for both ex vivo and in vivo administrations include suspensions (e.g., of genetically modified cells, liposomes, lipid nanoparticles or nanoparticles) in liquid or emulsified liquids. The active ingredients often are mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances, such as, wetting or emulsifying agents, pH buffering agents, stabilizing agents or other reagents that enhance the effectiveness of the pharmaceutical composition.
Applications
[0123] The methods and compositions disclosed herein are for providing therapies for any disease by provision of a transgene that expresses a product that is lacking or deficient in the disease or otherwise treats or prevents the disease. The cell may be modified in vivo or may be modified ex vivo and subsequently administered to a subject. Thus, the methods and compositions provide for the treatment and/or prevention of such genetic diseases. In addition, the methods and compositions disclosed herein allow for modification of B cells such that these cells exhibit modified toxicity, antibody production and/or processing characteristics.
[0124] The following Examples include exemplary embodiments of the present disclosure in which the optionally used nuclease comprises a zinc finger nuclease (ZFN). It will be appreciated that this is for purposes of exemplification only and that other nucleases can be used, for example TALENs, CRISPR/Cas systems, homing endonucleases (meganucleases) with engineered DNA-binding domains and/or fusions of naturally occurring of engineered homing endonucleases (meganucleases) DNA-binding domains and heterologous cleavage domains and/or fusions of meganucleases and TALE proteins. In addition, it will be appreciated that expression constructs as described herein can be carried on other vectors (other than AAV) to produce the same results in the treatment and/or prevention of disorders caused by deficient protein production.
EXAMPLES
Example 1: Methods
Cell Culture
[0125] Frozen human peripheral blood CD19+ B cells were purchased from STEMCELL Technologies (Vancouver, Canada). An in vitro B cell differentiation culture system (see
[0126] Cells were cultured in a 24-well plate at a density of 2.0E+5 cells per well in 0.5 mL of culture media. Cells were thawed and cultured for 4 days in B cell Activation Media containing Anti-His Ab (5 ?g/mL), ODN (10 ?g/mL), sCD40L (50 ng/mL), IL-2 (10 ng/mL), IL-10 (50 ng/mL), and IL-15 (10 ng/mL). At day 4 of culture, cells were harvested, supernatants were collected, cells were washed with DPBS and then transferred to Plasma Blast (PB) Generation Media containing IL-2 (10 ng/mL), IL-6 (40-50 ng/mL), IL-10 (50 ng/mL), and IL-15 (10 ng/mL). At day 7 of culture, cells were harvested, supernatants were collected, cells were washed DPBS and then transferred to Plasma Cell (PC) Generation Media containing IL-6 (40-50 ng/mL), IL-15 (10 ng/mL), IFN-? (500 U/mL). At day 10 of culture, cells were harvested and supernatants collected.
B Cell Gene Modification
ZFN Reagents:
[0127] ZFNs were designed to target TCRA (TRAC, SBS53909 and SBS53885, see U.S. Patent publication No. US-2017-0211075-A1), CCR5 (SBS8266 and SBS8196, see U.S. Pat. No. 7,925,921) and AAVS1 (SBS30035 and SBS30054, see U.S. Pat. No. 8,110,379). The CCR5 and AAVS1 ZFN coding sequences were cloned into a modified version of plasmid pGEM4Z (Promega, Madison, Wis.) containing a sequence of 64 adenines 3 of the inserted gene sequence (Boczkowski et al (2000) Canc Res 60:1028-1034), which was linearized by SpeI digestion to generate templates for mRNA synthesis. TRAC ZFN mRNA was produced from linear DNA templates (one for each ZFN) via PCR amplification of ZFN-encoding sequence with Accuprime PFX DNA Polymerase Kit (Invitrogen, Carlsbad, Calif.). PCR products were used as templates for mRNA synthesis. mRNA was prepared using the mMESSAGE mMACHINE T7 ULTRA Kit (Life Technologies, Carlsbad, Calif.) per manufacturer's protocol.
[0128] Briefly, 1.0 ?g of DNA encoding the ZFN was used as template for mRNA synthesis, incubated at 37? C. for two hours in supplied buffer, followed by DNAse digestion supplied with kit. The in vitro poly-A tailing reaction was not performed because a poly-T tail was incorporated on the DNA template during PCR generation of the TRAC template. The AAVS1 and CCR5 templates contain a poly-T template in the vector. mRNA was then purified using the RNeasy Mini Kit (Qiagen, Carlsbad, Calif.) per the manufacturer's protocol and quantified on the Nanodrop 8000 (ThermoScientific, Waltham, Mass.). The primers used for the mRNA templates were
TABLE-US-00001 ForwardPrimer: (SEQIDNO:1) 5 GCAGAGCTCTCTGGCTAACTAGAG and ReversePrimer: (SEQIDNO:2) 5 TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTTCTGGCAACTAGAAGGCACAG.
AAV Vectors:
[0129] All AAV vectors were produced at Sangamo Therapeutics as described below. AAV donor templates for AAVS1, CCR5 and TRAC contained homology arms to their target loci. AAVS1 had a left and right homology arm of 801 and 568 base pairs in length, respectively. CCR5 had a left and right homology arm of 473 and 1431 base pairs in length, respectively. TRAC had a left and right homology arm of 925 and 989 base pairs in length, respectively. A GFP expression cassette comprising a promoter, a GFP sequence and a human growth hormone polyadenylation signal (hGHpA) was cloned in between the right and left homology arms. The promoter was either a phosphoglycerate kinase (PGK) or B cell specific (EEK) promoter. The B cell specific (EEK) promoter consisted of a 3-enhancer, a MAR, and an intronic enhancer upstream of human ? light chain promoter (Luo et al (2009) Blood 113:1422-1431). The TRAC donor template was cloned into a pAAV vector. The AAVS1 and CCR5 donors templates were cloned into a customized plasmid pRS 165 (Lombardo et al (2011) Nat Methods 8:861-869; Wang et al (2012) Genome Res 22:1316-1326) derived from pAAV-MCS (Agilent Technologies, Santa Clara, Calif.). AAV2 inverted terminal repeats (ITRs) were used to enable packaging as AAV vectors using the triple-transfection method (Xiao and Samulski (1998) J. Virol 72:2224-2232). Briefly, HEK 293 cells were plated in 10-layer CellSTACK chambers (Corning, Acton, Mass.), grown for 3 days to a density of 80%, then transfected using the calcium phosphate method with an AAV helper plasmid expressing AAV2 Rep and serotype specific Cap genes, an adenovirus helper plasmid, and an ITR-containing donor vector plasmid. After 3 days the cells were lysed by three rounds of freeze/thaw, and cell debris removed by centrifugation. AAV vectors were precipitated from the lysates using polyethylene glycol, and purified by ultracentrifugation overnight on a cesium chloride gradient. Vectors were formulated by dialysis and filter sterilized.
IgM, IgG, IgA ELISA:
[0130] Supernatants were collected at the end of B cell activation, Plasma Blast Generation, and Plasma Cell Generation culture steps. IgM, IgG, IgA were assayed using commercial enzyme-linked immunosorbent assay (ELISA) kits (Bethyl Laboratories; Montgomery, Tex.) according to the manufacture's protocol. Briefly, supernatant was added to the plate, incubated with rocking at room temperature for one hour, followed by washing four times with buffer provided in the kit. Detecting antibody provided with the kit was added and incubated for 1 hour at room temperature, followed by washing four times with wash buffer provided in the kit. Horseradish peroxidase (HRP) provided with the kit was added and incubated for 30 minutes at room temperature, followed by washing four times with buffer provided in the kit. Tetramethylbenzidine (TMB) substrate provided with kit was added and allowed to develop for 30 minutes. The reaction was stopped with Stop Solution provided with the kit and absorbance read at 450 nM using a plate reader.
Example 2: Antibody Production in In Vitro Cultured B Cells
[0131] The CD19+ B cells were thawed and cultured as described above and illustrated in
[0132] The results (
Example 3: mRNA Electroporation
[0133] The cultured B cells were treated with mRNAs encoding a transgene (GFP) to determine the best time frame for introduction of the mRNA. The CD19+ B cells (2.0E+05 cells) were electroporated with 2 ?g GFP mRNA at days t0, t1, t2 or t3, where t0 is the day the cells were thawed (
Example 4: Nuclease Cleavage of the Cultured B Cells
[0134] ZFNs specific for three loci, AAVS1, CCR5 and TCRA were used to cleave their targets in the cultured B cells. CD19+ B cells were thawed and cultured for 2 days in B cell Activation Media. The cells were washed 2 times with DPBS then resuspended in BTXpress high performance electroporation solution (Harvard Apparatus, Holliston, Mass.) to a final density of 2.0E+6 cells/mL. Cells (100 ?L) and electroporation solution were mixed with ZFN mRNA (4 ?g) followed by electroporation in a BTX ECM830 Square Wave electroporator (Harvard Apparatus) in a MOS 96 multi-well Electroporation Plate 2 mm (Harvard Apparatus). Following electroporation cells were transferred to B cell Activation Media in a 24 well plate for two days. After 2 days, cells were harvested, supernatants were collected and cells were washed with DPBS then transferred to Plasma Blast Generation Media. After 3 days, cells were harvested, supernatants were collected and cells were washed with DPBS then transferred to Plasma Cells Generation Media. Cells were collected for genomic DNA (gDNA) isolation at days t4, t7 and t10 for ZFN activity analysis by deep sequencing. In brief, CD19+ B cells (2.0E+5 cells) were mixed with ZFN mRNA (4 ?g) followed by electroporation.
[0135] To measure ZFN activity at the TCRA (TRAC), CCR5, and AAVS1 loci, DNA was isolated by QIAamp DNA mini Kit (Qiagen, Carlsbad, Calif.) per the manufacturer's instructions. One hundred nanograms of genomic DNA (gDNA) was used. A two-step PCR for AAVS1 and TRAC loci was then carried out using Phusion? Hot Start Flex Polymerase (New England Biolabs, Ipswich, Mass.). A three-step PCR was used for CCR5 loci. Illumina deep sequencing measured indels at each loci. The primers used for each locus are shown below:
TABLE-US-00002 AAVS1Primers: AAVS1Forward: (SEQIDNO:3) GACGTGTGCTCTTCCGATCTNNNNCCGGTTAATGTGGCTCTGGT AAVS1Reverse: (SEQIDNO:4) ACACGACGCTCTTCCGATCTNNNNGACTAGGAAGGAGGAGGCCT. TheAAVS1ampliconwas: (SEQIDNO:5) 5NNNNGACTAGGAAGGAGGAGGCCTAAGGATGGGGCTTTTCTGTCAC CAATCCTGTCCCTAGTGGCCCCACTGTGGGGTGGAGGGGACAGATAAA AGTACCCAGAACCAGAGCCACATTAACCGGNNNN. CCR5Primers: CCR5Forward1: (SEQIDNO:6) CTGTGCTTCAAGGTCCTTGTCTGC, CCR5Reverse1: (SEQIDNO:7) CTCTGTCTCCTTCTACAGCCAAGC, CCR5Forward2: (SEQIDNO:8) CTGCCTCATAAGGTTGCCCTAAG, CCR5Reverse2: (SEQIDNO:9) CCAGCAATAGATGATCCAACTCAAATTCC, CCR5Forward3: (SEQIDNO:10) ACACGACGCTCTTCCGATCTNNNNNGCCAGGTTGAGCAGGTAGATG, CCR5Reverse3: (SEQIDNO:11) AGACGTGTGCTCTTCCGATCTGCTCTACTCACTGGTGTTCATCTTT. TheCCR5ampliconwas: (SEQIDNO:12) 5NNNNNGCCAGGTTGAGCAGGTAGATGTCAGTCATGCTCTTCAGCCT TTTGCAGTTTATCAGGATGAGGATGACCAGCATGTTGCCCACAAAACC AAAGATGAACACCAGTGAGTAGAGC. TCRA(TRAC)primers: TCRAForward: (SEQIDNO:13) 5ACACGACGCTCTTCCGATCTNNNNCCTCTTGGTTTTACAGATACGA AC TCRAReverse: (SEQIDNO:14) 5GACGTGTGCTCTTCCGATCTCTCACCTCAGCTGGACCAC TheTCRAampliconwas: (SEQIDNO:15) 5NNNNCCTCTTGGTTTTACAGATACGAACCTAAACTTTCAAAACCTG TCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAAT CTGCTCATGACGCTGCGGCTGTGGTCCAGCTGAGGTGAG.
[0136] The results of these studies are shown in
[0137] The experiments were also done testing the impact of a transient (overnight) cold shock (see U.S. Pat. No. 8,772,008) on the cleavage activity of the nucleases. In these studies, a range of input mRNA quantities were used from 0.75 to 6 ?g. After electroporation, the cultures were divided and one portion was placed in a 37? C. incubator for 4 days. The second group was placed in a 30? C. overnight, then transferred to a 37? C. incubator for 3 days. Deep sequencing was performed to measure the % indels detected as a result of the nuclease cleavage.
[0138] The results (as shown in
Example 5: B Cell AAV Serotype Transduction Comparison
[0139] AAV virus comprising a transgene (GFP) expression cassette were used to compare the ability of different AAV serotypes to transduce the cultured B cells. In brief, cells were thawed and cultured for 2 days in B cell Activation Media in a 24-well plate at a density of 2.0E+5 cells/well. Cells were collected, counted and then plated in a 24-well plate at a density of 2.0E+5 cells/well. B cells were transduced with AAV serotypes 2, 5, 6, 8 and 9 at vector doses of 2.4E+6, 1.2E+6, 6.0E+5, 3.0E+5 vector genomes (vg)/cell. AAV vector genomes contained CMV promoter-driven eGFP expression cassette and inverted terminal repeats (ITRs), see
[0140] The results (
Example 6: Nuclease Driven Targeted Integration
[0141] The nucleases described above were then used in combination with a transgene donor (GFP, proteins lacking or deficient in a subject and/or therapeutic antibodies of interest) to test the ability of the system to support targeted integration of a donor into the genome. Several exemplary donors were made with GFP (
[0142] CD19+ B cells were thawed and cultured for 2 days in B cell Activation Media. A combination of ZFN mRNA and AAV donor or mRNA donor targeting the same loci (AAVS1, TCRA, CCR5, albumin, HPRT, etc.) was used. The cells were washed 2 times with DPBS then resuspended in BTXpress high performance electroporation solution (Harvard Apparatus, Holliston, Mass.) to a final density of 2.0E+6 cells/mL. Cells (100 ?L) and electroporation solution were mixed with ZFN mRNA (4 ?g) followed by electroporation in a BTX ECM830 Square Wave electroporator (Harvard Apparatus) in a MOS 96 multi-well Electroporation Plate 2 mm (Harvard Apparatus). Following electroporation cells were transferred to 0.5 mL of B cell Activation Media in a 24 well plate. AAV containing homologous donor templates for target loci was then added at 2.4?10.sup.6 vg/cell. Plates were gently rocked for 2 minutes. After 2 days, cell culture was harvested, 25 ?L of cell culture was collected, mixed with DPBS (175 ?L) for flow cytometry analysis, the remaining cell culture was spun down in a table top centrifuge, supernatants collected, and cells washed with DPBS before being transferred to Plasma Blast Generation Media. After 3 days, cell culture was harvested, 25 ?L of cell culture was collected, mixed with DPBS (175 ?L) for flow cytometry analysis, the remaining cell culture was spun down in a table top centrifuge, supernatants collected and cells washed with DPBS before being transferred to Plasma Cells Generation Media. After 3 days the experiment concluded, cell culture (25 ?L from the 500 ?L in a single well of a 24-well plate) was collected and mixed with PBS (175 ?L) for flow cytometry analysis, the remaining cell culture was spun down in a table top centrifuge, supernatants collected, cells washed with DPBS and harvested for gDNA.
[0143] For the flow cytometry, cell culture (25 ?L from the 500 ?L in a single well of a 24-well plate) was collected and mixed with PBS (175 ?L) at days 2, 5 and 8 following the administration of mRNA and AAV donor. GFP expression was analyzed using a Guava EasyCyte 5HT (EMD Millipore, Billerica, Mass., USA). The data was analyzed using InCyte version 2.5 (EMD Millipore). The results (
[0144] To measure target integration of CCR5 and AAVS1 donors, DNA was isolated by a QIAamp DNA mini Kit (Qiagen, Carlsbad Calif.) per the manufacturer's instructions. One hundred nanograms of gDNA was used and a three-step PCR was then carried out using Phusion? Hot Start Flex Polymerase (New England Biolabs, Ipswich, Mass.) and HotStartTaq Master Mix Kit (Qiagen, Carlsbad, Calif.). Illumina deep sequencing measured target integration at each loci. The primers for each step are shown below:
TABLE-US-00003 AASV1Primers: Step1PCRPrimers: AAVS1Forward1: (SEQIDNO:16) 5CGGAACTCTGCCCTCTAACG. AAVS1Reverse1: (SEQIDNO:17) 5GTGTGTCACCAGATAAGGAATCTG. Step2PCRPrimers: AAVS1Forward2: (SEQIDNO:18) 5CGTCTCTCTCCTGAGTCCG. AAVS1Reverse2: (SEQIDNO:17) 5GTGTGTCACCAGATAAGGAATCTG. Step3PCRprimers: AAVS1Forward3: (SEQIDNO:19) 5CTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCTCTGGTTCTGG GTACTTTTATCTG. AAVS1Reverse3: (SEQIDNO:20) 5AGACGTGTGCTCTTCCGATCTGTGTGTCACCAGATAAGGAATCTG. AAVS1wildtypeampliconsequence: (SEQIDNO:21) 5NNNNCTCTGGTTCTGGGTACTTTTATCTGTCCCCTCCACCCCACAG TGGGGCCACTAGGGACAGGATTGGTGACAGAAAAGCCCCATCCTTAGG CCTCCTCCTTCCTAGTCTCCTGATATTGGGTCTAACCCCCACCTCCTG TTAGGCAGATTCCTTATCTGGTGACACAC. AAVS1GFP-TIsequence(SEQIDNO:22): 5NNNNCTCTGGTTCTGGGTACTTTTATCTGTCCCCTCCACCCCACAG TGGGGCAAGCTTCGAGCCATCAGGGCCTGGTTCTTTCCGCCTCAGAAG GCCTTTTGCAGTTTATCAGGATGAGGATGACCAGCATGTTGCCCACAA AACCAAAGATGAACACCAGATTCCTTATCTGGTGACACAC CCR5Primers Step1PCRPrimers: CCR5Forward1: (SEQIDNO:12) 5GCTCTACTCACTGGTGTTCATCTTT. CCR5Reverse1: (SEQIDNO:7) 5CTCTGTCTCCTTCTACAGCCAAGC. Step2PCRPrimers: CCR5Forward2: (SEQIDNO:12) 5GCTCTACTCACTGGTGTTCATCTTT. CCR5Reverse2: (SEQIDNO:9) 5CCAGCAATAGATGATCCAACTCAAATTCC. Step3PCRPrimers: CCR5Forward3: (SEQIDNO:23) 5ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNGCCAGGTT GAGCAGGTAGATG. CCR5Reverse3: (SEQIDNO:11) 5AGACGTGTGCTCTTCCGATCTGCTCTACTCACTGGTGTTCATCTT T. CCR5ampliconwildtypesequence: (SEQIDNO:12) 5NNNNNGCCAGGTTGAGCAGGTAGATGTCAGTCATGCTCTTCAGCCT TTTGCAGTTTATCAGGATGAGGATGACCAGCATGTTGCCCACAAAACC AAAGATGAACACCAGTGAGTAGAGC CCR5TI-GFPsequence: (SEQIDNO:24) 5NNNNNGCCAGGTTGAGCAGGTAGATGTCAGTCATGCTCTTCAGCCT TTTGCAGTTTCTCGAGCCATCAGGGCCTGGTTCTTTCCGCCTCAGAAG TAGAAAGATGAACACCAGTGAGTAGAGC
[0145] The results (as shown in
[0146] Experiments were also performed using non-matching transgene donors. The homology arms in these non-matching donors did not have homology with sequences flanking the nuclease target site of the co-introduced nuclease. For example, TCRA (TRAC)-specific ZFNs were used in combination with a GFP transgene donor comprising CCR5 homology arms. This donor was also used in combination with AAVS1-specific ZFN. Increased integration found with a matching ZFN target site and donor homology arms would indicate that, at least in part, the transgene integration relies on a homology dependent recombination reaction. The results (
[0147] The cells transduced with donor constructs comprising the two alternate promoters were also compared. GFP expression was analyzed by flow cytometry as described above (see
[0148] A titration comparing varying amounts of the AAV-donor construct was carried out using a constant dose of ZFN mRNA. The culture B cells were treated with 4? of TCRA (TRAC)-specific ZFN by electroporation, and then transduced with a range of donor AAV, from 3.0E+05 to 2.4E+06 vg/cell. Furthermore, the two promoters were also compared under these conditions. The results (
Example 7: Antibody Expression During Genome Editing
[0149] IgG and IgM levels were analyzed by ELISA as described above for the cultured B cells that had undergone electroporation for delivery of the ZFN pairs and also GFP donor. The results (
Example 8: Potential Booster Function of AAV in Cultured B Cells
[0150] In a cultured B cell that expresses an anti-AAV antibody, it could be possible that re-exposure of that B cell to the AAV serotype that the B cell is reactive against could cause a booster effect and induce an increase in anti-AAV antibody production. One CD19+ B cell population from one human donor demonstrated an increase in IgM secretion following treatment with AAV2. Anti-AAV2 antibodies are known to have a robust prevalence in the human population due to the ubiquitous presence of AAV2. Thus, in this study a CD19+ B cell population from one human donor potentially expressing anti-AAV2 antibodies was shown to induce a spike in IgM production following exposure to AAV2, but not other AAV serotypes (
[0151] A potential mechanism for the antibody expression spike is shown in
Example 9: Boost of Transgene Expression Following AAV2 Exposure In Vivo
[0152] Transgene donor cassettes are constructed for insertion of a transgene downstream of a B cell promoter. The B cells are treated ex vivo with a specific nuclease, and a donor construct comprising an antibody specific promoter linked to a transgene of interest. B cells chosen for this work are previously verified to produce anti-AAV2 antibodies. The cells are reintroduced into a subject and after a short period of time for engraftment, the subject is treated with AAV2, or AAV2 peptides. The AAV boost upregulates the antibody promoter causing a spike in transgene expression.
Example 10: B Cell Modification by Targeted Integration of B Cell-Specific Antibody
[0153] Transgene donor cassettes (AAV, mRNA, plasmid, etc.) are constructed for insertion of an antibody-encoding transgenes in which the antibody is(are) specific for B cell producing undesirable antibodies (e.g. inhibitors) against a protein delivered by ERT (or an autoantigen), for example B cell producing antibodies against a clotting factor such as F9 (anti-F9 antibodies). Donor cassettes can include homology arms to nuclease target loci (e.g., albumin, TCRA, CCR5, AAVS1, etc.) and are administered in vivo in combination with the suitable nuclease and/or ex vivo to B cell populations (mature, stem and/or B-cell progenitor cell populations) to a subject in need thereof (hemophilia patient with anti-F9 antibodies).
[0154] After ex vivo or in vivo modification, the antibody-producing B cells secrete the targeted antibodies which bind to the B cells producing the undesirable antibodies. These targeted antibodies then mediate lysis through mobilization and activation of antibody-dependent cytotoxic cells or though complement mediated lysis. Thus, in the patient these introduced B cells cause a reduction in the endogenous B cells that are producing undesirable antibodies for example, against proteins delivered by ERT or the autoantigen.
[0155] All patents, patent applications and publications mentioned herein are hereby incorporated by reference in their entirety.
[0156] Although disclosure has been provided in some detail by way of illustration and example for the purposes of clarity of understanding, it will be apparent to those skilled in the art that various changes and modifications can be practiced without departing from the spirit or scope of the disclosure. Accordingly, the foregoing descriptions and examples should not be construed as limiting.