Patent Application
20210340496
Regulatory T cells (Treg cells) play a role in regulating the immune response. In some cases, for example, in some cancers, Treg cells inhibit the ability of the immune system to target and destroy cancer cells. In other cases, for example in autoimmune diseases, Treg cells are unavailable to control the immune system. Methods to stabilize Treg cells for the treatment of autoimmune diseases or actively destabilize Treg cells to ablate tolerogenic effects in a tumor microenvironment have great therapeutic potential.
The present invention is directed to compositions and methods for modifying Treg cells. The inventors have identified nuclear factors that influence expression of Foxp3, a key transcriptional regulator of Treg cells. Treg cells can be modified by inhibiting and/or overexpressing one or more of these nuclear factors to produce stabilized Treg cells or destabilized Treg cells. In some examples, stabilized Treg cells are used to treat autoimmune disorders, assist in organ transplantation, to treat graft versus host disease, or inflammation. Examples of autoimmune diseases include but are not limited to: type 1 diabetes, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, and multi-organ autoimmune syndromes. In other examples, destabilized Treg cells are used to treat cancer. For example, in some embodiments, destabilized Tregs can be used to target solid tumors, e.g., where Treg cells contribute to a immunosuppressive microenvironment. Examples of such cancers include but are not limited to ovarian cancer.
Provided herein is a method of increasing human regulatory T (Treg) cell stability, the method comprising: inhibiting expression of a nuclear factor set forth in Table 1 and/or overexpressing a nuclear factor set forth in Table 2 in the human Treg cell.
Also provided is a method of decreasing human Treg cell stability is provided, the method comprising: inhibiting expression of a nuclear factor set forth in Table 2 and/or overexpressing a nuclear factor set forth in Table 1 in the human Treg cell.
In some embodiments, the inhibiting comprises reducing expression of a nuclear factor, or reducing expression of a polynucleotide encoding the nuclear factor in a Treg cell. In some embodiments, the overexpressing comprises increasing expression of a nuclear factor, or increasing expression of a polynucleotide encoding the nuclear factor in a Treg cell.
In some embodiments, the inhibiting in a Treg cell comprises contacting a polynucleotide encoding the protein with a targeted nuclease, a guide RNA (gRNA), an siRNA, an antisense RNA, microRNA (miRNA), or short hairpin RNA (shRNA). In some embodiments, the inhibiting comprises contacting the polynucleotide encoding the nuclear factor with at least one gRNA and optionally a targeted nuclease, wherein the at least one gRNA comprises a sequence selected from Table 3. In some embodiments, the inhibiting comprises mutating the polynucleotide encoding the protein. In some embodiments, the inhibiting comprises contacting the polynucleotide with a targeted nuclease.
In some embodiments, the targeted nuclease introduces a double-stranded break in a target region in the polynucleotide. In some embodiments, the targeted nuclease is an RNA-guided nuclease. In some embodiments, the RNA-guided nuclease is a Cpf1 nuclease or a Cas9 nuclease and the method further comprises introducing into a Treg cell a gRNA that specifically hybridizes to a target region in the polynucleotide. In some embodiments, the Cpf1 nuclease or the Cas9 nuclease and the gRNA are introduced into the Treg cell as a ribonucleoprotein (RNP) complex. In some embodiments, the inhibiting comprises performing clustered regularly interspaced short palindromic repeats (CRISPR)/Cas genome editing.
In some embodiments, the Treg cell is administered to a human following the inhibiting and/or the overexpressing. In some embodiments, the Treg cell is obtained from a human prior to treating the Treg cell to inhibit expression of the nuclear factor and/or overexpress the nuclear factor, and the treated Treg cell is reintroduced into a human. In some embodiments, expression of a nuclear factor is inhibited and/or a nuclear factor is overexpressed in an in vivo Treg cell. In some embodiments, the human has an autoimmune disorder, GVHD, inflammation, or is an organ transplantation recipient. In some embodiments, the human has cancer.
In another embodiment, provided herein is a Treg cell made by any of the methods described herein. In another embodiment, the present invention provides a Treg cell comprising a genetic modification or heterologous polynucleotide that inhibits expression of a nuclear factor selected set forth in Table 1 and/or a heterologous polynucleotide that encodes a protein encoded by a nuclear factor set forth in Table 2. In another embodiment, the present invention provides a Treg cell comprising a genetic modification or heterologous polynucleotide that inhibits expression of a nuclear factor set forth in Table 2 and/or a heterologous polynucleotide that encodes a polypeptide encoded by a nuclear factor set forth in Table 1.
In another embodiment, provided herein is a Treg comprising at least one guide RNA (gRNA) comprising a sequence selected from Table 3. In some embodiments, the expression of a nuclear factor set forth in Table 1 or Table 2 is reduced in the Treg cell relative to the expression of the nuclear factor in a Treg cell not comprising a gRNA.
In another embodiment, provided herein is a method of destabilizing Tregs in a subject in need thereof, comprising inhibiting expression of a one or more nuclear factors set forth in Table 2 and/or overexpressing one or more nuclear factors set forth in Table 1, in the humanTreg cells of the subject. In some embodiments, the Treg cells are destabilized in vivo. In other embodiments, the Treg cells are destabilized ex vivo. In some embodiments, the subject has cancer.
In another embodiment, provided herein is a method of stabilizing Tregs in a subject in need thereof, comprising inhibiting expression of a one or more nuclear factors set forth in Table 1 and/or overexpressing one or more nuclear factors set forth in Table 2, in the humanTreg cells of the subject. In some embodiments, the Treg cells are stabilized in vivo. In other embodiments, the Treg cells are stabilized ex vivo. In some embodiments, the subject has an autoimmune disorder.
In another embodiment, provided herein is a method of treating an autoimmune disorder in a subject, the method comprising administering a population of stabilized Treg cells to a subject that has an autoimmune disease. In another embodiment, the present invention provides a method of treating cancer in a subject, the method comprising administering a population of destabilized Treg cells to a subject that has cancer.
In another embodiment, provided herein is a method of treating an autoimmune disorder, GVHD, or inflammation, or assisting in organ transplantation treatment in a subject, the method comprising: (a) obtaining Treg cells from the subject (e.g., that has an autoimmune disorder); (b) modifying the Treg cells by inhibiting expression of a nuclear factor set forth in Table 1 and/or overexpressing a nuclear factor set forth in Table 2 in the Treg cells; and (c) administering the modified Treg cells to the subject.
In another embodiment, the present invention provides a method of treating cancer in a subject, the method comprising: (a) obtaining Treg cells from a subject that has cancer; (b) modifying the Treg cells by inhibiting expression of a nuclear factor set forth in Table 2 and/or overexpressing a nuclear factor set forth in Table 1 in the Treg cells; and (c) administering the modified Treg cells to the subject.
The present application includes the following figures. The figures are intended to illustrate certain embodiments and/or features of the compositions and methods, and to supplement any description(s) of the compositions and methods. The figures do not limit the scope of the compositions and methods, unless the written description expressly indicates that such is the case.
As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.
The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
The term “gene” can refer to the segment of DNA involved in producing or encoding a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
“Polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
The term “inhibiting expression” refers to inhibiting or reducing the expression of a gene product, e.g., RNA or protein. As used throughout, the term “nuclear factor” refers to a protein that directly or indirectly alters expression of Foxp3, for example, a transcription factor. To inhibit or reduce the expression of a gene, the sequence and/or structure of the gene may be modified such that the gene would not be transcribed (for DNA) or translated (for RNA), or would not be transcribed or translated to produce a functional protein, for example, a polypeptide or protein encoded by a gene set forth in Table 1 or Table 2. Various methods for inhibiting or reducing expression are described in detail further herein. Some methods may introduce nucleic acid substitutions, additions, and/or deletions into the wild-type gene. Some methods may also introduce single or double strand breaks into the gene. To inhibit or reduce the expression of a protein, one may inhibit or reduce the expression of the gene or polynucleotide encoding the protein. In other embodiments, one may target the protein directly to inhibit or reduce the protein's expression using, e.g., an antibody or a protease. “Inhibited” expression refers to a decrease by at least 10% as compared to a reference control level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. absent level as compared to a reference sample).
The term “overexpressing” or “overexpression” refers to increasing the expression of a gene or protein. “Overexpression” refers to an increase in expression, for example, in increase in the amount of mRNA or protein expressed in a Treg cell, of at least 10%, as compared to a reference control level, or an increase of least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 200%, or at least about 300% or at least about 400%. Various methods for overexpression are known to those of skill in the art, and include, but are not limited to, stably or transiently introducing a heterologous polynucleotide encoding a protein (i.e., a nuclear factor set forth in Table 1 or Table 2) to be overexpressed into the cell or inducing overexpression of an endogenous gene encoding the protein in the cell.
As used herein the phrase “heterologous” refers to what is not found in nature. The term “heterologous sequence” refers to a sequence not normally found in a given cell in nature. As such, a heterologous nucleotide or protein sequence may be: (a) foreign to its host cell (i.e., is exogenous to the cell); (b) naturally found in the host cell (i.e., endogenous) but present at an unnatural quantity in the cell (i.e., greater or lesser quantity than naturally found in the host cell); or (c) be naturally found in the host cell but positioned outside of its natural locus.
“Treating” refers to any indicia of success in the treatment or amelioration or prevention of the disease, condition, or disorder, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating.
A “promoter” is defined as one or more a nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
As used herein, the term “complementary” or “complementarity” refers to specific base pairing between nucleotides or nucleic acids. Complementary nucleotides are, generally, A and T (or A and U), and G and C. The guide RNAs described herein can comprise sequences, for example, DNA targeting sequences that are perfectly complementary or substantially complementary (e.g., having 1-4 mismatches) to a genomic sequence.
As used throughout, by subject is meant an individual. For example, the subject is a mammal, such as a primate, and, more specifically, a human Non-human primates are subjects as well. The term subject includes domesticated animals, such as cats, dogs, etc., livestock (for example, cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (for example, ferret, chinchilla, mouse, rabbit, rat, gerbil, guinea pig, etc.). Thus, veterinary uses and medical uses and formulations are contemplated herein. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. As used herein, patient or subject may be used interchangeably and can refer to a subject afflicted with a disease or disorder.
As used throughout, the term “targeted nuclease” refers to nuclease that is targeted to a specific DNA sequence in the genome of a cell to produce a strand break at that specific DNA sequence. The strand break can be single-stranded or double-stranded. Targeted nucleases include, but are not limited to, a Cas nuclease, a TAL-effector nuclease and a zinc finger nuclease.
The “CRISPR/Cas” system refers to a widespread class of bacterial systems for defense against foreign nucleic acid. CRISPR/Cas systems are found in a wide range of eubacterial and archaeal organisms. CRISPR/Cas systems include type I, II, and III sub-types. Wild-type type II CRISPR/Cas systems utilize an RNA-mediated nuclease, for example, Cas9, in complex with guide and activating RNA to recognize and cleave foreign nucleic acid. Guide RNAs having the activity of both a guide RNA and an activating RNA are also known in the art. In some cases, such dual activity guide RNAs are referred to as a single guide RNA (sgRNA).
Cas9 homologs are found in a wide variety of eubacteria, including, but not limited to bacteria of the following taxonomic groups: Actinobacteria, Aquificae, Bacteroidetes-Chlorobi, Chlamydiae-Verrucomicrobia, Chlroflexi, Cyanobacteria, Firmicutes, Proteobacteria, Spirochaetes, and Thermotogae. An exemplary Cas9 protein is the Streptococcus pyogenes Cas9 protein. Additional Cas9 proteins and homologs thereof are described in, e.g., Chylinksi, et al., RNA Biol. 2013 May 1; 10(5): 726-737; Nat. Rev. Microbiol. 2011 June; 9(6): 467-477; Hou, et al., Proc Natl Acad Sci USA. 2013 Sep. 24; 110(39):15644-9; Sampson et al., Nature. 2013 May 9; 497(7448):254-7; and Jinek, et al., Science. 2012 Aug. 17; 337(6096):816-21. Variants of any of the Cas9 nucleases provided herein can be optimized for efficient activity or enhanced stability in the host cell. Thus, engineered Cas9 nucleases are also contemplated.
As used throughout, a guide RNA (gRNA) sequence is a sequence that interacts with a site-specific or targeted nuclease and specifically binds to or hybridizes to a target nucleic acid within the genome of a cell, such that the gRNA and the targeted nuclease co-localize to the target nucleic acid in the genome of the cell. Each gRNA includes a DNA targeting sequence or protospacer sequence of about 10 to 50 nucleotides in length that specifically binds to or hybridizes to a target DNA sequence in the genome. For example, the targeting sequence may be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the gRNA comprises a crRNA sequence and a transactivating crRNA (tracrRNA) sequence. In some embodiments, the gRNA does not comprise a tracrRNA sequence. Table 3 shows exemplary gRNA sequences used in methods of the disclosure.
As used herein, the term “Cas9” refers to an RNA-mediated nuclease (e.g., of bacterial or archeal orgin, or derived therefrom). Exemplary RNA-mediated nucleases include the foregoing Cas9 proteins and homologs thereof. Other RNA-mediated nucleases include Cpf1 (See, e.g., Zetsche et al., Cell, Volume 163, Issue 3, p′759-′7′71, 22 Oct. 2015) and homologs thereof. Similarly, as used herein, the term “Cas9 ribonucleoprotein” complex and the like refers to a complex between the Cas9 protein and a guide RNA, the Cas9 protein and a crRNA, the Cas9 protein and a trans-activating crRNA (tracrRNA), or a combination thereof (e.g., a complex containing the Cas9 protein, a tracrRNA, and a crRNA guide RNA). It is understood that in any of the embodiments described herein, a Cas9 nuclease can be subsitututed with a Cpf1 nuclease or any other guided nuclease.
As used herein, the phrase “modifying” refers to inducing a structural change in the sequence of the genome at a target genomic region in a Treg cell. For example, the modifying can take the form of inserting a nucleotide sequence into the genome of the cell. Such modifying can be performed, for example, by inducing a double stranded break within a target genomic region, or a pair of single stranded nicks on opposite strands and flanking the target genomic region. Methods for inducing single or double stranded breaks at or within a target genomic region include the use of a Cas9 nuclease domain, or a derivative thereof, and a guide RNA, or pair of guide RNAs, directed to the target genomic region. “Modifying” can also refer to altering the expression of a nuclear factor in a Treg cell, for example inhibiting expression of a nuclear factor or overexpressing a nuclear factor in a Treg cell.
As used herein, the phrase “introducing” in the context of introducing a nucleic acid or a complex comprising a nucleic acid, for example, an RNP complex, refers to the translocation of the nucleic acid sequence or the RNP complex from outside a cell to inside the cell. In some cases, introducing refers to translocation of the nucleic acid or the complex from outside the cell to inside the nucleus of the cell. Various methods of such translocation are contemplated, including but not limited to, electroporation, contact with nanowires or nanotubes, receptor mediated internalization, translocation via cell penetrating peptides, liposome mediated translocation, and the like.
The following description recites various aspects and embodiments of the present compositions and methods. No particular embodiment is intended to define the scope of the compositions and methods. Rather, the embodiments merely provide non-limiting examples of various compositions and methods that are at least included within the scope of the disclosed compositions and methods. The description is to be read from the perspective of one of ordinary skill in the art; therefore, information well known to the skilled artisan is not necessarily included.
Treg cells are a specialized subset of CD4+ T cells that suppress inflammation to maintain homeostasis and prevent autoimmunity. Treg cell development and function depend on expression of the master transcription factor Foxp3. While Treg cells have been thought to be irreversibly committed to suppressive functions, lineage tracing studies have revealed that Treg cells can exhibit plasticity. Treg cells that lose Foxp3 expression, termed ‘exTregs’, have been shown to acquire cytokine production capabilities of pro-inflammator effector T cells and exacerbate autoimmunity. However, the gene regulatory programs that promote or disrupt Foxp3 stability in Treg cells under various physiological conditions are not well understood. The inventors have identified nuclear factors that regulate expression of Foxp3, thereby altering Treg cell stability.
As described herein, the disclosure provides compositions and methods directed to modifying regulatory T (Treg) cell stability by inhibiting the expression of one or more nuclear factors and/or overexpressing one or more nuclear factors in a Treg cell. The disclosure also features compositions comprising the Treg cells having modified stability. A population of modified Treg cells that are destabilized may provide therapeutic benefits in treating cancer. A population of modified Treg cells that are stabilized may provide therapeutic benefits in treating autoimmune diseases.
The present disclosure is directed to compositions and methods for modifying the stability of regulatory T cells (also referred to as “Treg cells”). The inventors have discovered that by inhibiting the expression of one or more nuclear factors and/or overexpressing one or more nuclear factors, the stability of Treg cells may be altered. In some embodiments, the Treg cells may be destabilized by inhibiting the expression of one or more nuclear factors and/or overexpressing one or more nuclear factors, such that they may have less immunosuppressive effects and improved therapeutic benefits towards treating cancer. A population of destabilized Treg cells may be used to enhance or improve various cancer therapies or Treg cells of an individual having cancer can be targeted to destabilize the Treg cells. In other embodiments, Treg cells may be stabilized by inhibiting the expression of one or more nuclear factors and/or overexpressing one or more nuclear factors, such that they may have more immunosuppressive effects and improved therapeutic benefits towards treating an autoimmune disease. A population of stabilized Treg cells may be used to treat or alleviate autoimmune diseases or Treg cells of an individual having an autoimmune disease can be targeted to stabilize the Treg cells.
Examples of nuclear factors whose expression may be altered to modify the stability of Treg cells in the methods described herein include, but are not limited to the nuclear factors set forth in Table 1 and Table 2. In some embodiments, the present invention provides a method of increasing regulatory T (Treg) cell stability, the method comprising: inhibiting expression of one or more nuclear factors set forth in Table 1 and/or overexpressing one or more nuclear factors set forth in Table 2 in the Treg cell. Inhibition of one or more nuclear factors set forth in Table 1 and/or overexpression of one or more nuclear factors set forth in Table 2 may increase Foxp3 expression in the Treg cell or stabilize Foxp3 expression (e.g., in an inflammatory environment that would otherwise result in Foxp3 expression reduction), thereby increasing stability of the Treg cell.
In other embodiments, the present invention provides a method of decreasing Treg cell stability, the method comprising: inhibiting expression of one or more nuclear factors set forth in Table 2 and/or overexpressing one or more nuclear factors set forth in Table 1, in the Treg cell. Inhibition of one or more nuclear factors set forth in Table 2 and/or overexpression of one or more nuclear factors set forth in Table 1 may decrease Foxp3 expression in the Treg cell, thereby decreasing stability of the Treg cell. Table 1 provides nuclear factors that, when inhibited, increase Foxp3 expression. Overexpression of a nuclear factor set forth in Table 1 may decrease Foxp3 expression. In some embodiments, expression of an amino acid sequence having at least about 80%, 85%, 90%, 95% or 99% identity to an amino acid sequence set forth in Table 1 is inhibited. In some embodiments, an amino acid sequence having at least about 80%, 85%, 90%, 95% or 99% identity to an amino acid sequence set forth in Table 1 is overexpressed. Table 2 provides nuclear factors that, when inhibited, decrease Foxp3 expression. Overexpression of a nuclear factor set forth in Table 2 may increase Foxp3 expression. In some embodiments, expression of an amino acid sequence having at least about 80%, 85%, 90%, 95% or 99% identity to an amino acid sequence set forth in Table 2 is inhibited. In some embodiments, an amino acid sequence having at least about 80%, 85%, 90%, 95% or 99% identity to an amino acid sequence set forth in Table 2 is overexpressed. It is understood that, when referring to one or more nuclear factors set forth in Table 1 or Table 2, this can be the protein, i.e., the nuclear factor, or the polynucleotide encoding the nuclear factor.
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Stability of Treg cells may be assessed using FACS markers. Some of the FACS markers used are canonical Treg cell signature proteins. For example, with a specific gene knocked-out or inhibited in Treg cells, if these modified cells display a gain or maintenance of Treg cell canonical markers, such as FOXP3, CTLA4, CD25, IL-10, and/or IKZF2, this may indicate the Treg cells are more stabilized. In some embodiments, a loss of Treg cell canonical markers and/or gain of pro-inflammatory markers (e.g., IL-17a, IL-4, IFNγ, and IL-2) may indicate that the Treg cells are destabilized. In another example, with overexpression of a specific nuclear factor in Treg cells, if these modified cells display a gain or maintenance of Treg cell canonical markers, such as FOXP3, CTLA4, CD25, IL-10, and/or IKZF2, this may indicate the Treg cells are more stabilized. In some embodiments, with overexpression of a specific nuclear factor in Treg cells, if these modified cells display a loss of Treg cell canonical markers and/or gain of pro-inflammatory markers (e.g., IL-17a, IL-4, IFNγ, and IL-2), this may indicate that the Treg cells are destabilized. For methods of detecting and enriching for Tregs, see, for example, International Patent Application Publication No. WO2007140472.
In some embodiments of the methods described herein, inhibiting the expression of a nuclear factor set forth in Table 1 or Table 2 may comprise reducing expression of the nuclear factor or reducing expression of a polynucleotide, for example, an mRNA, encoding the nuclear factor in the Treg cell. In some embodiments expression of one or more nuclear factor s set forth in Table 1 or Table 2 is inhibited in the Treg cell. As described in detail further herein, one or more available methods may be used to inhibit the expression of one or more nuclear factors set forth in Table 1 or Table 2.
In some embodiments of the methods described herein, overexpressing a nuclear factor set forth in Table 1 or a nuclear factor set forth in Table 2 may comprise introducing a polynucleotide encoding the nuclear factor into the Treg cell. In other embodiments of the methods described herein, overexpressing a nuclear factor set forth in Table 1 or a nuclear factor set forth in Table 2 may comprise introducing an agent that induces expression of the endogenous gene encoding the nuclear factor in the Treg cell. For example, RNA activation, where short double-stranded RNAs induce endogenous gene expression by targeting promoter sequences, can be used to induce endogenous gene expression (See, for example, Wang et al. “Inducing gene expression by targeting promoter sequences using small activating RNAs,” J. Biol. Methods 2(1): e14 (2015). In another example, artificial transcription factors containing zinc-finger binding domains can be used to activate or repress expression of endogenous genes. See, for example, Dent et al., “Regulation of endogenous gene expressing using small molecule-controlled engineered zinc-finger protein transcription factors,” Gene Ther. 14(18): 1362-9 (2007).
In some embodiments, inhibiting expression may comprise contacting a polynucleotide encoding the nuclear factor, with a target nuclease, a guide RNA (gRNA), an siRNA, an antisense RNA, microRNA (miRNA), or short hairpin RNA (shRNA). In particular embodiments, if a gRNA and a target nuclease (e.g., Cas9) are used to inhibit the expression of a polynucleotide encoding a human nuclear factor set forth in Table 1 or Table 2, the gRNA may comprise a sequence set forth in Table 3, a sequence complementary to a sequence set forth in Table 3, or a portion thereof. Table 3 provides the Gene ID number, Genbank Accession No. for mRNA, genomic sequence, position in the genome after nuclease cutting, sgRNA target sequence, target context sequence, PAM sequence, and the exon targeted by the sgRNA for each nuclear factor set forth in Tables 1 and 2. ZNF281 is the human homolog of mouse Zfp281.
As described herein, the stability of Treg cells may be modified by inhibiting the expression of the one or more nuclear factors set forth in Table 1 or Table 2. The stability of Treg cells may also be modified by overexpressing one or more nuclear factors set forth in Table 1 or Table 2. Subsequently, once modified Treg cells are created, the modified Treg cells may be administered to a human. Depending on whether the Treg cells are stabilized or destabilized, the modified Treg cells may be used to treat different indications. For example, Treg cells may be isolated from a whole blood sample of a human and expanded ex vivo. The expanded Treg cells may then be treated to inhibit the expression of a nuclear factor set forth in Table 1 or Table 2 thus, creating modified Treg cells. The modified Treg cells may be reintroduced to the human to treat certain indications. In some embodiments, destabilized Treg cells having less immunosuppressive effects may be used to treat cancer. In some embodiments, stabilized Treg cells having improved immunosuppressive effects may be used to treat autoimmune diseases. Certain nuclear factors in Treg cells increase Foxp3 expression (Table 1) and have a stabilizing effect once their expression is inhibited, while other nuclear factors decrease Foxp3 expression (Table 2) in Treg cells and have a destabilizing effect once their expression is inhibited. Cell stability may be determined by a multi-color FACS panel based on Treg cell markers like Foxp3, Helios, CTLA-4, CD25, IL-10, and effectors such as cytokines typically associated with effector T cell subsets like IL-2, IFNγ, IL-17a, and IL-4. Assays for measuring Treg cell stability can be found in, e.g., McClymont, et al., “Plasticity of Human Regulatory T Cells in Healthy Subjects and Patients with Type 1 Diabetes” J. immunol. 186 (2011). Depending on the indication and therapeutic needs, one may choose to target one or more nuclear factors to generate modified Treg cells that are destabilized or stabilized.
In other cases, Treg cells in a subject can be modified in vivo, for example, by using a targeted vector, such as, a lentiviral vector, a retroviral vector an adenoviral or adeno-associated viral vector. In vivo delivery of targeted nucleases that modify the genome of a Treg cell can also be used. See for example, U.S. Pat. No. 9,737,604 and Zhang et al. “Lipid nanoparticle-mediated efficient delivery of CRISPR/Cas9 for tumor therapy,” NPG Asia Materials Volume 9, page e441 (2017).
Also provided is a Treg cell wherein expression of one or more nuclear factors set forth in Table 1 or Table 2 is inhibited. Further provided is a Treg cell wherein one or more nuclear factors set forth in Table 1 or Table 2 is overexpressed. The disclosure also features a Treg cell comprising a genetic modification or heterologous polynucleotide that inhibits expression of one or more nuclear factors set forth in Table 1 and/or a heterologous polynucleotide that encodes a nuclear factor set forth in Table 2. Also provided is a Treg cell comprising a genetic modification or heterologous polynucleotide that inhibits expression of a nuclear factor set forth in Table 2 and/or a heterologous polynucleotide that encodes a nuclear factor set forth in Table 1.
A genetic modification may be a nucleotide mutation or any sequence alteration in the polynucleotide encoding the nuclear factor that results in the inhibition of the expression of the nuclear factor. A heterologous polynucleotide may refer to a polynucleotide originally encoding the nuclear factor but is altered, i.e., comprising one or more nucleotide mutations or sequence alterations. In some embodiments, the heterologous polynucleotide is inserted into the genome of the Treg cell by introducing a vector, for example, a viral vector, comprising the polynucleotide. Examples of viral vectors include, but are not limited to adeno-associated viral (AAV) vectors, retroviral vectors or lentiviral vectors. In some embodiments, the lentiviral vector is an integrase-deficient lentiviral vector.
Also disclosed herein are Treg cells comprising at least one guide RNA (gRNA) comprising a sequence selected from Table 3. The expression of one or more nuclear factors set forth in Table 1 or Table 2, in the Treg cells comprising the gRNAs, may be reduced in the Treg cells relative to the expression of the one or more nuclear factors in Treg cells not comprising the gRNAs. In other examples, an endogenous nuclear factor set forth in Table 1 or Table 2 can be inhibited by targeting a deactivated targeted nuclease, for example dCAs9, fused to a transcriptional repressor, to the promoter region of the endogenous nuclear factor gene. In other examples, an endogenous nuclear factor set forth in Table 1 or Table 2 can be upregulated or overexpressed by targeting a deactivated targeted nuclease, for example dCAs9, fused to a transcriptional activator, to the promoter region of the endogenous nuclear factor gene. See, for example, Qi et al. “The New State of the Art: Cas9 for Gene Activation and Repression,” Mol. and Cell. Biol., 35(22): 3800-3809 (2015).
CRISPR/Cas Genome Editing
The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated protein) nuclease system is an engineered nuclease system based on a bacterial system that can be used for genome engineering. It is based on part of the adaptive immune response of many bacteria and archaea. When a virus or plasmid invades a bacterium, segments of the invader's DNA are converted into CRISPR RNAs (crRNA) by the “immune” response. The crRNA then associates, through a region of partial complementarity, with another type of RNA called tracrRNA to guide the Cas (e.g., Cas9) nuclease to a region homologous to the crRNA in the target DNA called a “protospacer.” The Cas (e.g., Cas9) nuclease cleaves the DNA to generate blunt ends at the double-strand break at sites specified by a 20-nucleotide guide sequence contained within the crRNA transcript. The Cas (e.g., Cas9) nuclease can require both the crRNA and the tracrRNA for site-specific DNA recognition and cleavage. This system has now been engineered such that the crRNA and tracrRNA can be combined into one molecule (the “guide RNA” or “gRNA”), and the crRNA equivalent portion of the single guide RNA can be engineered to guide the Cas (e.g., Cas9) nuclease to target any desired sequence (see, e.g., Jinek et al. (2012) Science 337:816-821; Jinek et al. (2013) eLife 2:e00471; Segal (2013) eLife 2:e00563). Thus, the CRISPR/Cas system can be engineered to create a double-strand break at a desired target in a genome of a cell, and harness the cell's endogenous mechanisms to repair the induced break by homology-directed repair (HDR) or nonhomologous end-joining (NHEJ).
In some embodiments of the methods described herein, CRISPR/Cas genome editing may be used to inhibit the expression of one or more nuclear factors set forth in Table 1 or Table 2.
In some embodiments, the Cas nuclease has DNA cleavage activity. The Cas nuclease can direct cleavage of one or both strands at a location in a target DNA sequence, i.e., a location in a polynucleotide encoding a nuclear factor set forth in Table 1 or Table 2. In some embodiments, the Cas nuclease can be a nickase having one or more inactivated catalytic domains that cleaves a single strand of a target DNA sequence.
Non-limiting examples of Cas nucleases include Cast, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, variants thereof, mutants thereof, and derivatives thereof. There are three main types of Cas nucleases (type I, type II, and type III), and 10 subtypes including 5 type I, 3 type II, and 2 type III proteins (see, e.g., Hochstrasser and Doudna, Trends Biochem Sci, 2015:40(1):58-66). Type II Cas nucleases include Cas1, Cas2, Csn2, and Cas9. These Cas nucleases are known to those skilled in the art. For example, the amino acid sequence of the Streptococcus pyogenes wild-type Cas9 polypeptide is set forth, e.g., in NBCI Ref. Seq. No. NP_269215, and the amino acid sequence of Streptococcus thermophilus wild-type Cas9 polypeptide is set forth, e.g., in NBCI Ref. Seq. No. WP_011681470. Some CRISPR-related endonucleases that may be used in methods described herein are disclosed, e.g., in U.S. Application Publication Nos. 2014/0068797, 2014/0302563, and 2014/0356959.
Cas nucleases, e.g., Cas9 polypeptides, can be derived from a variety of bacterial species including, but not limited to, Veillonella atypical, Fusobacterium nucleatum, Filifactor alocis, Solobacterium moorei, Coprococcus catus, Treponema denticola, Peptoniphilus duerdenii, Catenibacterium mitsuokai, Streptococcus mutans, Listeria innocua, Staphylococcus pseudintermedius, Acidaminococcus intestine, Olsenella uli, Oenococcus kitaharae, Bifidobacterium bifidum, Lactobacillus rhamnosus, Lactobacillus gasseri, Finegoldia magna, Mycoplasma mobile, Mycoplasma gallisepticum, Mycoplasma ovipneumoniae, Mycoplasma canis, Mycoplasma synoviae, Eubacterium rectale, Streptococcus thermophilus, Eubacterium dolichum, Lactobacillus coryniformis subsp. Torquens, Ilyobacter polytropus, Ruminococcus albus, Akkermansia muciniphila, Acidothermus cellulolyticus, Bifidobacterium longum, Bifidobacterium dentium, Corynebacterium diphtheria, Elusimicrobium minutum, Nitratifractor salsuginis, Sphaerochaeta globus, Fibrobacter succinogenes subsp. Succinogenes, Bacteroides fragilis, Capnocytophaga ochracea, Rhodopseudomonas palustris, Prevotella micans, Prevotella ruminicola, Flavobacterium columnare, Aminomonas paucivorans, Rhodospirillum rubrum, Candidatus Puniceispirillum marinum, Verminephrobacter eiseniae, Ralstonia syzygii, Dinoroseobacter shibae, Azospirillum, Nitrobacter hamburgensis, Bradyrhizobium, Wolinella succinogenes, Campylobacter jejuni subsp. Jejuni, Helicobacter mustelae, Bacillus cereus, Acidovorax ebreus, Clostridium perfringens, Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria meningitidis, Pasteurella multocida subsp. Multocida, Sutterella wadsworthensis, proteobacterium, Legionella pneumophila, Parasutterella excrementihominis, Wolinella succinogenes, and Francisella novicida.
Wild-type Cas9 nuclease has two functional domains, e.g., RuvC and HNH, that cut different DNA strands. Cas9 can induce double-strand breaks in genomic DNA (target DNA) when both functional domains are active. The Cas9 enzyme can comprise one or more catalytic domains of a Cas9 protein derived from bacteria belonging to the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, and Campylobacter. In some embodiments, the Cas9 may be a fusion protein, e.g., the two catalytic domains are derived from different bacteria species.
Useful variants of the Cas9 nuclease can include a single inactive catalytic domain, such as a RuvC− or HNH− enzyme or a nickase. A Cas9 nickase has only one active functional domain and can cut only one strand of the target DNA, thereby creating a single strand break or nick. In some embodiments, the Cas9 nuclease may be a mutant Cas9 nuclease having one or more amino acid mutations. For example, the mutant Cas9 having at least a D10A mutation is a Cas9 nickase. In other embodiments, the mutant Cas9 nuclease having at least a H840A mutation is a Cas9 nickase. Other examples of mutations present in a Cas9 nickase include, without limitation, N854A and N863A. A double-strand break may be introduced using a Cas9 nickase if at least two DNA-targeting RNAs that target opposite DNA strands are used. A double-nicked induced double-strand break can be repaired by NHEJ or HDR (Ran et al., 2013, Cell, 154:1380-1389). This gene editing strategy favors HDR and decreases the frequency of INDEL mutations at off-target DNA sites. Non-limiting examples of Cas9 nucleases or nickases are described in, for example, U.S. Pat. Nos. 8,895,308; 8,889,418; and 8,865,406 and U.S. Application Publication Nos. 2014/0356959, 2014/0273226 and 2014/0186919. The Cas9 nuclease or nickase can be codon-optimized for the target cell or target organism.
In some embodiments, the Cas nuclease can be a Cas9 polypeptide that contains two silencing mutations of the RuvC1 and HNH nuclease domains (D10M and H840A), which is referred to as dCas9 (Jinek et al., Science, 2012, 337:816-821; Qi et al., Cell, 152(5):1173-1183). In one embodiment, the dCas9 polypeptide from Streptococcus pyogenes comprises at least one mutation at position D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, A987 or any combination thereof. Descriptions of such dCas9 polypeptides and variants thereof are provided in, for example, International Patent Publication No. WO 2013/176772. The dCas9 enzyme may contain a mutation at D10, E762, H983, or D986, as well as a mutation at H840 or N863. In some instances, the dCas9 enzyme may contain a D10A or DION mutation. Also, the dCas9 enzyme may contain a H840A, H840Y, or H840N. In some embodiments, the dCas9 enzyme may contain D10A and H840A; D10A and H840Y; D10A and H840N; D10N and H840A; D10N and H840Y; or D10N and H840N substitutions. The substitutions can be conservative or non-conservative substitutions to render the Cas9 polypeptide catalytically inactive and able to bind to target DNA.
In some embodiments, the Cas nuclease can be a high-fidelity or enhanced specificity Cas9 polypeptide variant with reduced off-target effects and robust on-target cleavage. Non-limiting examples of Cas9 polypeptide variants with improved on-target specificity include the SpCas9 (K855A), SpCas9 (K810A/K1003A/R1060A) (also referred to as eSpCas9(1.0)), and SpCas9 (K848A/K1003A/R1060A) (also referred to as eSpCas9(1.1)) variants described in Slaymaker et al., Science, 351(6268):84-8 (2016), and the SpCas9 variants described in Kleinstiver et al., Nature, 529(7587):490-5 (2016) containing one, two, three, or four of the following mutations: N497A, R661A, Q695A, and Q926A (e.g., SpCas9-HF1 contains all four mutations).
As described above, a gRNA may comprise a crRNA and a tracrRNAs. The gRNA can be configured to form a stable and active complex with a gRNA-mediated nuclease (e.g., Cas9 or dCas9). The gRNA contains a binding region that provides specific binding to the target genetic element. Exemplary gRNAs that may be used to target a region in a polynucleotide encoding a nuclear factor set forth in Table 1 or Table 2 are listed in Table 3 below. A gRNA used to target a region in a polynucleotide encoding a nuclear factor set forth in Table 1 or Table 2 may comprise a sequence selected from Table 3 below or a portion thereof.
In some embodiments, the targeted nuclease, for example, a Cpf1 nuclease or a Cas9 nuclease and the gRNA are introduced into the Treg cell as a ribonucleoprotein (RNP) complex. In some embodiments, the RNP complex may be introduced into about 1×105 to about 2×106 cells (e.g., 1×105 cells to about 5×105 cells, about 1×105 cells to about 1×106 cells, 1×105 cells to about 1.5×106 cells, 1×105 cells to about 2×106 cells, about 1×106 cells to about 1.5×106 cells, or about 1×106 cells to about 2×106 cells). In some embodiments, the Treg cells are cultured under conditions effective for expanding the population of modified Treg cells. Also disclosed herein is a population of Treg cells, in which the genome of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or greater of the cells comprises a genetic modification or heterologous polynucleotide that inhibits expression of one or more nuclear factors set forth in Table 1 or Table 2.
In some embodiments, the RNP complex is introduced into the Treg cells by electroporation. Methods, compositions, and devices for electroporating cells to introduce a RNP complex are available in the art, see, e.g., WO 2016/123578, WO/2006/001614, and Kim, J. A. et al. Biosens. Bioelectron. 23, 1353-1360 (2008). Additional or alternative methods, compositions, and devices for electroporating cells to introduce a RNP complex can include those described in U.S. Patent Appl. Pub. Nos. 2006/0094095; 2005/0064596; or 2006/0087522; Li, L. H. et al. Cancer Res. Treat. 1, 341-350 (2002); U.S. Pat. Nos. 6,773,669; 7,186,559; 7,771,984; 7,991,559; 6,485,961; 7,029,916; and U.S. Patent Appl. Pub. Nos: 2014/0017213; and 2012/0088842; Geng, T. et al., J. Control Release 144, 91-100 (2010); and Wang, J., et al. Lab. Chip 10, 2057-2061 (2010).
In some embodiments, the sequence of the gRNA or a portion thereof is designed to complement (e.g., perfectly complement) or substantially complement (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99% complement) the target region in the polynucleotide encoding the protein. In some embodiments, the portion of the gRNA that complements and binds the targeting region in the polynucleotide is, or is about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or more nucleotides in length. In some cases, the portion of the gRNA that complements and binds the targeting region in the polynucleotide is between about 19 and about 21 nucleotides in length. In some cases, the gRNA may incorporate wobble or degenerate bases to bind target regions. In some cases, the gRNA can be altered to increase stability. For example, non-natural nucleotides, can be incorporated to increase RNA resistance to degradation. In some cases, the gRNA can be altered or designed to avoid or reduce secondary structure formation. In some cases, the gRNA can be designed to optimize G-C content. In some cases, G-C content is between about 40% and about 60% (e.g., 40%, 45%, 50%, 55%, 60%). In some cases, the binding region can contain modified nucleotides such as, without limitation, methylated or phosphorylated nucleotides
In some embodiments, the gRNA can be optimized for expression by substituting, deleting, or adding one or more nucleotides. In some cases, a nucleotide sequence that provides inefficient transcription from an encoding template nucleic acid can be deleted or substituted. For example, in some cases, the gRNA is transcribed from a nucleic acid operably linked to an RNA polymerase III promoter. In such cases, gRNA sequences that result in inefficient transcription by RNA polymerase III, such as those described in Nielsen et al., Science. 2013 Jun. 28; 340(6140):1577-80, can be deleted or substituted. For example, one or more consecutive uracils can be deleted or substituted from the gRNA sequence. In some cases, if the uracil is hydrogen bonded to a corresponding adenine, the gRNA sequence can be altered to exchange the adenine and uracil. This “A-U flip” can retain the overall structure and function of the gRNA molecule while improving expression by reducing the number of consecutive uracil nucleotides.
In some embodiments, the gRNA can be optimized for stability. Stability can be enhanced by optimizing the stability of the gRNA:nuclease interaction, optimizing assembly of the gRNA:nuclease complex, removing or altering RNA destabilizing sequence elements, or adding RNA stabilizing sequence elements. In some embodiments, the gRNA contains a 5′ stem-loop structure proximal to, or adjacent to, the region that interacts with the gRNA-mediated nuclease. Optimization of the 5′ stem-loop structure can provide enhanced stability or assembly of the gRNA:nuclease complex. In some cases, the 5′ stem-loop structure is optimized by increasing the length of the stem portion of the stem-loop structure.
gRNAs can be modified by methods known in the art. In some cases, the modifications can include, but are not limited to, the addition of one or more of the following sequence elements: a 5′ cap (e.g., a 7-methylguanylate cap); a 3′ polyadenylated tail; a riboswitch sequence; a stability control sequence; a hairpin; a subcellular localization sequence; a detection sequence or label; or a binding site for one or more proteins. Modifications can also include the introduction of non-natural nucleotides including, but not limited to, one or more of the following: fluorescent nucleotides and methylated nucleotides.
Also described herein are expression cassettes and vectors for producing gRNAs in a host cell. The expression cassettes can contain a promoter (e.g., a heterologous promoter) operably linked to a polynucleotide encoding a gRNA. The promoter can be inducible or constitutive. The promoter can be tissue specific. In some cases, the promoter is a U6, H1, or spleen focus-forming virus (SFFV) long terminal repeat promoter. In some cases, the promoter is a weak mammalian promoter as compared to the human elongation factor 1 promoter (EF1A). In some cases, the weak mammalian promoter is a ubiquitin C promoter or a phosphoglycerate kinase 1 promoter (PGK). In some cases, the weak mammalian promoter is a TetOn promoter in the absence of an inducer. In some cases, when a TetOn promoter is utilized, the host cell is also contacted with a tetracycline transactivator. In some embodiments, the strength of the selected gRNA promoter is selected to express an amount of gRNA that is proportional to the amount of Cas9 or dCas9. The expression cassette can be in a vector, such as a plasmid, a viral vector, a lentiviral vector, etc. In some cases, the expression cassette is in a host cell. The gRNA expression cassette can be episomal or integrated in the host cell.
Zinc-Finger Nucleases (ZFNs)
“Zinc finger nucleases” or “ZFNs” are a fusion between the cleavage domain of FokI and a DNA recognition domain containing 3 or more zinc finger motifs. The heterodimerization at a particular position in the DNA of two individual ZFNs in precise orientation and spacing leads to a double-strand break in the DNA. In some embodiments of the methods described herein, ZFNs may be used to inhibit the expression of one or more nuclear factors set forth in Table 1 or Table 2, i.e., by cleaving the polynucleotide encoding the protein.
In some cases, ZFNs fuse a cleavage domain to the C-terminus of each zinc finger domain. In order to allow the two cleavage domains to dimerize and cleave DNA, the two individual ZFNs bind opposite strands of DNA with their C-termini at a certain distance apart. In some cases, linker sequences between the zinc finger domain and the cleavage domain requires the 5′ edge of each binding site to be separated by about 5-7 bp. Exemplary ZFNs that may be used in methods described herein include, but are not limited to, those described in Urnov et al., Nature Reviews Genetics, 2010, 11:636-646; Gaj et al., Nat Methods, 2012, 9(8):805-7; U.S. Pat. Nos. 6,534,261; 6,607,882; 6,746,838; 6,794,136; 6,824,978; 6,866,997; 6,933,113; 6,979,539; 7,013,219; 7,030,215; 7,220,719; 7,241,573; 7,241,574; 7,585,849; 7,595,376; 6,903,185; 6,479,626; and U.S. Application Publication Nos. 2003/0232410 and 2009/0203140.
ZFNs can generate a double-strand break in a target DNA, resulting in DNA break repair which allows for the introduction of gene modification. DNA break repair can occur via non-homologous end joining (NHEJ) or homology-directed repair (HDR). In HDR, a donor DNA repair template that contains homology arms flanking sites of the target DNA can be provided.
In some embodiments, a ZFN is a zinc finger nickase which can be an engineered ZFN that induces site-specific single-strand DNA breaks or nicks, thus resulting in HDR. Descriptions of zinc finger nickases are found, e.g., in Ramirez et al., Nucl Acids Res, 2012, 40(12):5560-8; Kim et al., Genome Res, 2012, 22(7):1327-33.
TALENs
TALENS may also be used to inhibit the expression of one or more nuclear factors set forth in Table 1 or Table 2. “TALENs” or “TAL-effector nucleases” are engineered transcription activator-like effector nucleases that contain a central domain of DNA-binding tandem repeats, a nuclear localization signal, and a C-terminal transcriptional activation domain. In some instances, a DNA-binding tandem repeat comprises 33-35 amino acids in length and contains two hypervariable amino acid residues at positions 12 and 13 that can recognize one or more specific DNA base pairs. TALENs can be produced by fusing a TAL effector DNA binding domain to a DNA cleavage domain. For instance, a TALE protein may be fused to a nuclease such as a wild-type or mutated FokI endonuclease or the catalytic domain of FokI. Several mutations to Fold have been made for its use in TALENs, which, for example, improve cleavage specificity or activity. Such TALENs can be engineered to bind any desired DNA sequence.
TALENs can be used to generate gene modifications by creating a double-strand break in a target DNA sequence, which in turn, undergoes NHEJ or HDR. In some cases, a single-stranded donor DNA repair template is provided to promote HDR.
Detailed descriptions of TALENs and their uses for gene editing are found, e.g., in U.S. Pat. Nos. 8,440,431; 8,440,432; 8,450,471; 8,586,363; and U.S. Pat. No. 8,697,853; Scharenberg et al., Curr Gene Ther, 2013, 13(4):291-303; Gaj et al., Nat Methods, 2012, 9(8):805-7; Beurdeley et al., Nat Commun, 2013, 4:1762; and Joung and Sander, Nat Rev Mol Cell Biol, 2013, 14(1):49.
Meganucleases
Meganucleases” are rare-cutting endonucleases or homing endonucleases that can be highly specific, recognizing DNA target sites ranging from at least 12 base pairs in length, e.g., from 12 to 40 base pairs or 12 to 60 base pairs in length. Meganucleases can be modular DNA-binding nucleases such as any fusion protein comprising at least one catalytic domain of an endonuclease and at least one DNA binding domain or protein specifying a nucleic acid target sequence. The DNA-binding domain can contain at least one motif that recognizes single- or double-stranded DNA. The meganuclease can be monomeric or dimeric.
In some embodiments of the methods described herein, meganucleases may be used to inhibit the expression of one or more nuclear factors set forth in Table 1 or Table 2 i.e., by cleaving in a target region within the polynucleotide encoding the nuclear factor. In some instances, the meganuclease is naturally-occurring (found in nature) or wild-type, and in other instances, the meganuclease is non-natural, artificial, engineered, synthetic, or rationally designed. In certain embodiments, the meganucleases that may be used in methods described herein include, but are not limited to, an I-CreI meganuclease, I-CeuI meganuclease, I-MsoI meganuclease, I-SceI meganuclease, variants thereof, mutants thereof, and derivatives thereof.
Detailed descriptions of useful meganucleases and their application in gene editing are found, e.g., in Silva et al., Curr Gene Ther, 2011, 11(1):11-27; Zaslavoskiy et al., BMC Bioinformatics, 2014, 15:191; Takeuchi et al., Proc Natl Acad Sci USA, 2014, 111(11):4061-4066, and U.S. Pat. Nos. 7,842,489; 7,897,372; 8,021,867; 8,163,514; 8,133,697; 8,021,867; 8,119,361; 8,119,381; 8,124,36; and 8,129,134.
RNA-Based Technologies
Various RNA-based technologies may also be used in methods described herein to inhibit the expression of one or more nuclear factors set forth in Table 1 or Table 2. Examples of RNA-based technologies include, but are not limited to, small interfering RNA (siRNA), antisense RNA, microRNA (miRNA), and short hairpin RNA (shRNA).
RNA-based technologies may use an siRNA, an antisense RNA, a miRNA, or a shRNA to target a sequence, or a portion thereof, that encodes a transcription factor. In some embodiments, one or more genes regulated by a transcription factor may also be targeted by an siRNA, an antisense RNA, a miRNA, or a shRNA. An siRNA, an antisense RNA, a miRNA, or a shRNA may target a sequence comprising at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 contiguous nucleotides.
An siRNA may be produced from a short hairpin RNA (shRNA). A shRNA is an artificial RNA molecule with a hairpin turn that can be used to silence target gene expression via the siRNA it produces in cells. See, e.g., Fire et. al., Nature 391:806-811, 1998; Elbashir et al., Nature 411:494-498, 2001; Chakraborty et al., Mol Ther Nucleic Acids 8:132-143, 2017; and Bouard et al., Br. J. Pharmacol. 157:153-165, 2009. Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors. Suitable bacterial vectors include but not limited to adeno-associated viruses (AAVs), adenoviruses, and lentiviruses. After the vector has integrated into the host genome, the shRNA is then transcribed in the nucleus by polymerase II or polymerase III (depending on the promoter used). The resulting pre-shRNA is exported from the nucleus, then processed by a protein called Dicer and loaded into the RNA-induced silencing complex (RISC). The sense strand is degraded by RISC and the antisense strand directs RISC to an mRNA that has a complementary sequence. A protein called Ago2 in the RISC then cleaves the mRNA, or in some cases, represses translation of the mRNA, leading to its destruction and an eventual reduction in the protein encoded by the mRNA. Thus, the shRNA leads to targeted gene silencing.
The shRNA or siRNA may be encoded in a vector. In some embodiments, the vector further comprises appropriate expression control elements known in the art, including, e.g., promoters (e.g., inducible promoters or tissue specific promoters), enhancers, and transcription terminators.
Any of the methods described herein may be used to modify Treg cells in a human subject or obtained from a human subject. Any of the methods and compositions described herein may be used to modify Treg cells obtained from a human subject to treat or prevent a disease (e.g., cancer, an autoimmune disease, an infectious disease, transplantation rejection, graft vs. host disease or other inflammatory disorder in a subject).
Provided herein is a method of treating an autoimmune disorder in a subject, the method comprising administering a population of Treg cells comprising a genetic modification or heterologous polynucleotide that inhibits expression of a nuclear factor set forth in Table 1 and/or a heterologous polynucleotide that encodes a nuclear factor set forth in Table 2 to a subject that has an autoimmune disorder.
Also provided is a method of treating cancer in a subject, the method comprising administering a population of Treg cells comprising a genetic modification or heterologous polynucleotide that inhibits expression of a nuclear factor set forth in Table 2 and/or a heterologous polynucleotide that encodes a nuclear factor set forth in Table 1 to a subject that has cancer.
Provided herein is a method of treating cancer in a human subject comprising: a) obtaining Treg cells from the subject; b) modifying the Treg cells using any of the methods provided herein to decrease the stability of the Treg cells; and c) administering the modified Treg cells to the subject, wherein the human subject has cancer. Also provided herein is a method of treating an autoimmune disease in a human subject comprising: a) obtaining Treg cells from the subject; b) modifying the Treg cells using any of the methods provided herein to increase the stability of the Treg cells; and c) administering the modified Treg cells to the subject, wherein the human subject has an autoimmune disease.
In some embodiments, Treg cells obtained from a cancer subject may be expanded ex vivo. The characteristics of the subject's cancer may determine a set of tailored cellular modifications (i.e., which nuclear factors from Table 1 and/or Table 2 to target), and these modifications may be applied to the Treg cells using any of the methods described herein. Modified Treg cells may then be reintroduced to the subject. This strategy capitalizes on and enhances the function of the subject's natural repertoire of cancer specific T cells, providing a diverse arsenal to eliminate mutagenic cancer cells quickly. Similar strategies may be applicable for the treatment of autoimmune diseases, in which the modified Treg cells would have improved stability.
In other cases, Treg cells in a subject can be targeted for in vivo modification. See, for example, See, for example, U.S. Pat. No. 9,737,604 and Zhang et al. “Lipid nanoparticle-mediated efficient delivery of CRISPR/Cas9 for tumor therapy,” NPG Asia Materials Volume 9, page e441 (2017).
Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed and a number of modifications that can be made to one or more molecules including in the method are discussed, each and every combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.
Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties.
The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially the same or similar results.
B6 Foxp3-GFP-Cre mice (Zhou et al., “Selective miRNA disruption in T reg cells leads to uncontrolled autoimmunity,” J Exp Med. 205, 1983-91 (2008)) were crossed with B6 Rosa26-RFP reporter mice (Luche et al., “Faithful activation of an extra-bright red fluorescent protein in “knock-in” Cre-reporter mice ideally suited for lineage tracing studies,” Eur. J. Immunol. 37, 43-53 (2007)) as previously described (Bailey-Bucktrout et al., “Self-antigen-driven activation induces instability of regulatory T cells during an inflammatory autoimmune response, Immunity. 39, 949-62 (2013)) to generate the Foxp3 fate reporter mice (
Spleens and peripheral lymph nodes were harvested from mice and dissociated in 1×PBS with 2% FBS and 1 mM EDTA. The mixture was then passed through a 70-μm filter. CD4+ T cells were isolated using the CD4+ Negative Selection Kit (StemCell Technologies, Cat #19752) followed by fluorescence-activated cell sorting. For the prescreen sort, Tregs were gated on lymphocytes, live cells, CD4+, CD62L+, RFP+, Foxp3-GFP+ cells. For the arrayed validation experiments, Tregs were gated on lymphocytes, live cells, CD4+, Foxp3-GFP+ cells. Sorted Tregs were cultured in complete DMEM, 10% FBS, 1% pen/strep+2000U hIL-2 in 24 well plates at 1 million cells/mL. Tregs were stimulated using CD3/CD28 Mouse T-Activator Dynabeads (Thermo Fisher, Cat #11456D) at a ratio of 3:1 beads to cells for 48 hours. Cells were split and media was refreshed every 2-3 days.
Pooled sgRNA Library Design and Construction
For the cloning of the targeted library, we followed the custom sgRNA library cloning protocol as previously described (Joung et al., “Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening,” Nat Protocols. 12, 828-863 (2017)). We utilized a MSCV-U6-sgRNA-IRES-Thy1.1 backbone. To optimize this plasmid for cloning the library, we first replaced the sgRNA with a 1.9 kb stuffer derived from the lentiGuide-Puro plasmid (Addgene, plasmid #52963) with flanking BsgI cut sites. This stuffer was excised using the BsgI restriction enzyme (NEB, Cat #R0559) and the linear backbone was gel purified (Qiagen, Cat #28706). We designed a targeted library to include all genes matching Gene Ontology for “Nucleic Acid Binding Transcription Factors”, “Protein Binding Transcription Factors”, “Involved in Chromatin Organization” and “Involved in Epigenetic Regulation”. Genes were then selected based on those that have the highest expression levels across any mouse CD4 T cell subset as defined by Stubbington et al. (Stubbington et al., “An atlas of mouse CD4+ T cell transcriptomes,” Biol Direct. 10. 14 (2015)). In total, we included 493 targets with 4 guides per gene, and 28 non-targeting controls. Guides were subsetted from the Brie sgRNA library (Doench et al., “Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9,” Nature biotechnology. 34 (2), 184-191 (2016)), and the pooled oligo library was ordered from Twist Bioscience to match the vector backbone. Oligos were PCR amplified and cloned into the modified MSCV backbone by Gibson assembly as described by Joung et al. The library was amplified using Endura ElectroCompetent Cells following the manufacturer's protocol (Endura, Cat #60242-1).
Platinum-E (Plat-E) Retroviral Packaging cells (Cell Biolabs, Inc., Cat #RV-101) were seeded at 10 million cells in 15 cm poly-L-Lysine coated dishes 16 hours prior to transfection and cultured in complete DMEM, 10% FBS, 1% pen/strep, 1 μg/mL puromycin and 10 μg/mL blasticidin Immediately before transfection, the media was replaced with antibiotic free complete DMEM, 10% FBS. The cells were transfected with the sgRNA transfer plasmids (MSCV-U6-sgRNA-IRES-Thy1.1) using TransIT-293 transfection reagent per the manufacturer's protocol (Mirus, Cat #MIR 2700). The following morning, the media was replaced with complete DMEM, 10% FBS, 1% pen/strep. The viral supernatant was collected 48 hours post-transfection and filtered through a 0.45 μm, polyethersulfone sterile syringe filter (Whatman, Cat #6780-2504), to remove cell debris. The viral supernatant was aliquoted and stored until use at −80° C.
Tregs were stimulated as described above for 48-60 hours. Cells were counted and seeded at 3 million cells in 1 mL of media with 2×hIL-2 into each well of a 6 well plate that was coated with 15 μg/mL of RetroNectin (Takara, Cat #T100A) for 3 hours at room temperature and subsequently washed with 1×PBS. Retrovirus was added at a 1:1 v/v ratio (1 mL) and plates were centrifuged for 1 hour at 2000 g at 30° C. and placed in the incubator at 37° C. overnight. The next day, half (1 mL) of the 1:1 retrovirus to media mixture was removed from the plate and 1 mL of fresh retrovirus was added. Plates were immediately centrifuged for 1 hour at 2000 g at 30° C. After the second spinfection, cells were pelleted, washed, and cultured in fresh media.
Tregs were collected from their culture vessels 8 days after the second transduction and centrifuged for 5 min at 300 g. Cells were first stained with a viability dye at a 1:1,000 dilution in 1×PBS for 20 min at 4° C., then washed with EasySep Buffer (1×PBS, 2% FBS, 1 mM EDTA). Cells were then resuspended in the appropriate surface staining antibody cocktail and incubated for 30 min at 4° C., then washed with EasySep Buffer. Cells were then fixed, permeabilized, and stained for transcription factors using the Foxp3 Transcription Factor Staining Buffer Set (eBioscience, Cat #00-5523-00) according to the manufacturer's instructions. For the CRISPR screen, Foxp3 high and Foxp3 low populations were isolated using fluorescence-activated cell sorting by gating on lymphocytes, live cells, CD4+ and gating on the highest 40% of Foxp3-expressing cells (Foxp3 high) and lowest 40% of Foxp3-expressing cells (Foxp3 low) by endogenous Foxp3 intracellular staining. Over 2 million cells were collected for both sorted populations to maintain a library coverage of at least 1,000 cells per sgRNA.
Isolation of Genomic DNA from Fixed Cells
After cell sorting and collection, genomic DNA (gDNA) was isolated using a protocol specific for fixed cells. Cell pellets were resuspended in cell lysis buffer (0.5% SDS, 50 mM Tris, pH 8, 10 mM EDTA) with 1:25 v/v of 5M NaCl to reverse crosslinking and incubated at 66° C. overnight. RNase A (10 mg/mL) was added at 1:50 v/v and incubated at 37° C. for 1 hour. Proteinase K (20 mg/mL) was added at 1:50 v/v and incubated at 45° C. for 1 hour. Phenol:Chloroform:Isoamyl Alcohol (25:24:1) was added to the sample 1:1 v/v and transferred to a phase lock gel light tube (QuantaBio, Cat #2302820), inverted vigorously and centrifuged at 20,000 g for 5 mins. The aqueous phase was then transferred to a clean tube and NaAc at 1:10 v/v, 1 μl of GeneElute-LPA (Sigma, Cat #56575), and isopropanol at 2.5:1 v/v were added. The sample was vortexed, and incubated at −80° C. until frozen solid. Then thawed and centrifuged at 20,000 g for 30 mins. The cell pellet was washed with 500 μl of 75% EtOH, gently inverted and centrifuged at 20,000 g for 5 mins, aspirated, dried, and resuspended in 20 μl TE buffer.
Amplification and bar-coding of sgRNAs for the cell surface sublibrary was performed as previously described (Gilbert et al., “Genome scale CRISPR-mediated control of gene repression and activation,’ Cell. 159, 647-661 (2014)) with some modifications. Briefly, after gDNA isolation, sgRNAs were amplified and barcoded with TruSeq Single Indexes using a one-step PCR. TruSeq Adaptor Index 12 (CTTGTA) was used for the Foxp3 low population and TrueSeq Adaptor Index 14 (AGTTCC) was used for the Foxp3 high population. Each PCR reaction consisted of 50 μL of NEBNext Ultra II Q5 Master Mix (NEB #M0544), 1 μg of gDNA, 2.5 μL each of the 10 μM forward and reverse primers, and water to 1004, total. The PCR cycling conditions were: 3 minutes at 98° C., followed by 10 seconds at 98° C., 10 seconds at 62° C., 25 seconds at 72° C., for 26 cycles; and a final 2 minute extension at 72° C. After the PCR, the samples were purified using Agencourt AMPure XPSPRI beads (Beckman Coulter, cat #A63880) per the manufacturer's protocol, quantified using the Qubit ssDNA high sensitivity assay kit (Thermo Fisher Scientific, cat #Q32854), and then analyzed on the 2100 Bioanalyzer Instrument. Samples were then sequenced on an Illumina MiniSeq using a custom sequencing primer.
Primary Tregs were isolated from the spleen and lymph nodes of three male Foxp3-GFP-Cre/Rosa26-RFP/Cas9 mice aged 5-7 months old, pooled together, and stimulated for 60 hours. Cells were then retrovirally transduced with the sgRNA library and cultured at a density of 1 million cells/ml continually maintaining a library coverage of at least 1,000 cells per sgRNA. Eight days after the second transduction, cells were sorted based on Foxp3 expression defined by intracellular staining. Genomic DNA was harvested from each population and the sgRNA-encoding regions were then amplified by PCR and sequenced on an Illumina MiniSeq using custom sequencing primers. From this data, we quantified the frequencies of cells expressing different sgRNAs in each in each population (Foxp3 high and Foxp3 low) and quantified the phenotype of the sgRNAs, which we have defined as Foxp3 stabilizing (enriched in Foxp3 high) or Foxp3 destabilizing (enriched in Foxp3 low) (
Analysis was performed as previously described (Shifrut et al., “Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function. Biorxiv. (2018)doi: https://doi.org/10.1101/384776)). To identify hits from the screen, we used the MAGeCK software to quantify and test for guide enrichment (Li et al., “MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens,” Genome Biol. 15, 554 (2014)). Abundance of guides was first determined by using the MAGeCK “count” module for the raw fastq files. For the targeted libraries the constant 5′ trim was automatically detected by MAGeCK. To test for robust guide and gene-level enrichment, the MAGeCK “test” module was used with default parameters. This step includes median ratio normalization to account for varying read depths. We used the non-targeting control guides to estimate the size factor for normalization, as well as to build the mean-variance model for null distribution, which is used to find significant guide enrichment. MAGeCK produced guide-level enrichment scores for each direction (i.e. positive and negative) which were then used for alpha-robust rank aggregation (RRA) to obtain gene-level scores. The p-value for each gene is determined by a permutation test, randomizing guide assignments and adjusted for false discovery rates by the Benjamini-Hochberg method. Log 2 fold change (LFC) is also calculated for each gene, defined throughout as the median LFC for all guides per gene target. Where indicated, LFC was normalized to have a mean of 0 and standard deviation of 1 to obtain the LFC Z-score.
RNPs were produced by complexing a two-component gRNA to Cas9, as previously described (Schumann et al., “Generation of knock-in primary human T cells using Cas9 ribonucleoproteins,” Proc. Natl Acad. Sci. USA. 112, 10437-10442 (2015)). In brief, crRNAs and tracrRNAs were chemically synthesized (IDT), and recombinant Cas9-NLS were produced and purified (QB3 Macrolab). Lyophilized RNA was resuspended in Nuclease-free Duplex Buffer (IDT, Cat #1072570) at a concentration of 160 μM, and stored in aliquots at −80° C. crRNA and tracrRNA aliquots were thawed, mixed 1:1 by volume, and annealed by incubation at 37° C. for 30 min to form an 80 μM gRNA solution. Recombinant Cas9 was stored at 40 μM in 20 mM HEPES-KOH, pH 7.5, 150 mM KCl, 10% glycerol, 1 mM DTT, were then mixed 1:1 by volume with the 80 μM gRNA (2:1 gRNA to Cas9 molar ratio) at 37° C. for 15 min to form an RNP at 20 μM. RNPs were electroporated immediately after complexing. RNPs were electroporated 3 days after initial stimulation, Tregs were collected from their culture vessels and centrifuged for 5 min at 300 g, aspirated, and resuspended in the Lonza electroporation buffer P3 using 20 μl buffer per 200,000 cells. 200,000 Tregs were electroporated per well using a Lonza 4D 96-well electroporation system with pulse code EO148. Immediately after electroporation, 80 μL of pre-warmed media was added to each well and the cells were incubated at 37° C. for 15 minutes. The cells were then transferred to a round-bottom 96-well tissue culture plate and cultured in complete DMEM, 10% FBS, 1% pen/strep+2000U hIL-2 at 200,000 cells/well in 200 μl of media.
Primary human Treg cells for all experiments were obtained from residuals from leukoreduction chambers after Trima Apheresis (Blood Centers of the Pacific) under a protocol approved by the UCSF Committee on Human Research (CHR #13-11950). Peripheral blood mononuclear cells (PBMCs) were isolated from samples by Lymphoprep centrifugation (StemCell, Cat #07861) using SepMate tubes (StemCell, Cat #85460). CD4+ T cells were isolated from PBMCs by magnetic negative selection using the EasySep Human CD4+ T Cell Isolation Kit (StemCell, Cat #17952) and Tregs were then isolated using fluorescence-activated cell sorting by gating on CD4+, CD25+, CD127low cells. After isolation, cells were stimulated with ImmunoCult Human CD3/CD28/CD2 T Cell Activator (StemCell, Cat #10970) per the manufacturer's protocol and expanded for 9 days. Cells were cultured in complete RPMI media, 10% FBS, 50 mM 2-mercaptoethanol and 1% pen/strep with hIL-2 at 300 U/mL at 1 million cells/mL. After expansion, Tregs were restimulated in the same way for 24 h before RNP electroporation.
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Additional studies were conducted to validate the role of previously undescribed candidate genes from the CRISPR screen including Rnf20 and members of the SAGA deubiquitination module, Usp22 and Atxn713. CRISPR-Cas9 ribonucleoproteins (RNP) were used to knock out candidate genes in both human and mouse primary Tregs and changes were identified in several Treg characteristic markers and pro-inflammatory cytokines by flow cytometry. Five of the top-ranking positive regulators were assessed by invidual CRISPR knockout with Cas9 RNPs. All guides tested resulted in a decrease in Foxp3 expression reproducing the screen data (
It was also found that Usp22 and Atxn713 knockouts in mouse Tregs reduces Foxp3 expression (
This application claims the benefit of U.S. Provisional Application No. 62/744,058, filed on Oct. 10, 2018, which is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/055674 | 10/10/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62744058 | Oct 2018 | US |
