COMPOSITIONS AND METHODS FOR TREATING OR PREVENTING CROHN'S DISEASE
20220370509 · 2022-11-24
Inventors
Cpc classification
C12N2310/20
CHEMISTRY; METALLURGY
A61P1/04
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
C12N9/22
CHEMISTRY; METALLURGY
C12N2740/16043
CHEMISTRY; METALLURGY
C12N2750/14143
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
A61P1/02
HUMAN NECESSITIES
C12N15/113
CHEMISTRY; METALLURGY
A61K48/005
HUMAN NECESSITIES
A61K9/0019
HUMAN NECESSITIES
A61K35/15
HUMAN NECESSITIES
A61P1/00
HUMAN NECESSITIES
A61K35/545
HUMAN NECESSITIES
A61K48/0066
HUMAN NECESSITIES
A61K35/28
HUMAN NECESSITIES
C12N15/86
CHEMISTRY; METALLURGY
International classification
A61K35/545
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K48/00
HUMAN NECESSITIES
Abstract
Described herein are compositions and methods for treating a subject having or at risk of developing Crohn's disease. Using the compositions and methods of the disclosure, a patient, such as an adult human patient, may be provided one or more agents that elevate the expression and/or activity levels of Nucleotide-binding oligomerization domain-containing protein 2 (NOD2). Exemplary agents that may be used in conjunction with the compositions and methods of the disclosure for this purpose include cells, such as pluripotent cells, that express NOD2.
Claims
1. A method of treating Crohn's disease in a patient in need thereof, the method comprising providing to the patient one or more agents that increase expression and/or activity of Nucleotide-binding oligomerization domain-containing protein 2 (NOD2).
2. A method of inducing sustained disease remission of Crohn's disease in a patient in need thereof, the method comprising providing to the patient one or more agents that increase expression and/or activity of NOD2.
3. A method of increasing muramyl dipeptide (MDP) sensing in a patient that has Crohn's disease, the method comprising providing to the patient one or more agents that increase expression and/or activity of NOD2.
4. A method of increasing NFκB signal transduction detection in a patient that has Crohn's disease, the method comprising providing to the patient one or more agents that increase expression and/or activity of NOD2.
5. The method of any one of claims 1-4, wherein the one or more agents comprise a nucleic acid molecule that encodes NOD2.
6. The method of claim 5, wherein the nucleic acid molecule is provided to the patient by administering to the patient a composition comprising a population of cells that express NOD2.
7. The method of claim 6, wherein the cells are pluripotent cells.
8. The method of claim 6 or 7, wherein the cells are human cells.
9. The method of any one of claims 6-8, wherein the cells are hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs).
10. The method of any one of claims 6-8, wherein the cells are embryonic stem cells.
11. The method of any one of claims 6-8, wherein the cells are induced pluripotent stem cells.
12. The method of any one of claims 6-8, wherein the cells are CD34+ cells.
13. The method of claim 12, wherein the CD34+ cells are myeloid progenitor cells.
14. The method of any one of claims 6-13, wherein the composition is administered systemically to the patient.
15. The method of claim 14, wherein the composition is administered to the patient by way of intravenous injection.
16. The method of any one of claims 6-15, wherein the cells are autologous with respect to the patient.
17. The method of any one of claims 6-15, wherein the cells are allogeneic with respect to the patient.
18. The method of claim 17, wherein the cells are HLA-matched to the patient.
19. The method of any one of claims 6-18, wherein the cells are transduced ex vivo to express NOD2.
20. The method of claim 19, wherein the cells are transduced with a viral vector selected from the group consisting of a Retroviridae family virus, an adenovirus, a parvovirus, a coronavirus, a rhabdovirus, a paramyxovirus, a picornavirus, an alphavirus, a herpes virus, and a poxvirus.
21. The method of claim 20, wherein the viral vector is a Retroviridae family viral vector.
22. The method of claim 21, wherein the Retroviridae family viral vector is a lentiviral vector.
23. The method of claim 21, wherein the Retroviridae family viral vector is an alpharetroviral vector or a gammaretroviral vector.
24. The method of any one of claims 20-23, wherein the Retroviridae family viral vector comprises a central polypurine tract, a woodchuck hepatitis virus post-transcriptional regulatory element, a 5′-LTR, HIV signal sequence, HIV Psi signal 5′-splice site, delta-GAG element, 3′-splice site, and a 3′-self inactivating LTR.
25. The method of any one of claims 20-24, wherein the viral vector is a pseudotyped viral vector.
26. The method of claim 25, wherein the pseudotyped viral vector selected from the group consisting of a pseudotyped adenovirus, a pseudotyped parvovirus, a pseudotyped coronavirus, a pseudotyped rhabdovirus, a pseudotyped paramyxovirus, a pseudotyped picornavirus, a pseudotyped alphavirus, a pseudotyped herpes virus, a pseudotyped poxvirus, and a pseudotyped Retroviridae family virus.
27. The method of claim 25 or 26, wherein the pseudotyped viral vector comprises one or more envelope proteins from a virus selected from vesicular stomatitis virus (VSV), RD114 virus, murine leukemia virus (MLV), feline leukemia virus (FeLV), Venezuelan equine encephalitis virus (VEE), human foamy virus (HFV), walleye dermal sarcoma virus (WDSV), Semliki Forest virus (SFV), Rabies virus, avian leukosis virus (ALV), bovine immunodeficiency virus (BIV), bovine leukemia virus (BLV), Epstein-Barr virus (EBV), Caprine arthritis encephalitis virus (CAEV), Sin Nombre virus (SNV), Cherry Twisted Leaf virus (ChTLV), Simian T-cell leukemia virus (STLV), Mason-Pfizer monkey virus (MPMV), squirrel monkey retrovirus (SMRV), Rous-associated virus (RAV), Fujinami sarcoma virus (FuSV), avian carcinoma virus (MH2), avian encephalomyelitis virus (AEV), Alfa mosaic virus (AMV), avian sarcoma virus CT10, and equine infectious anemia virus (EIAV).
28. The method of claim 27, wherein the pseudotyped viral vector comprises a VSV-G envelope protein.
29. The method of any one of claims 6-18, wherein the cells are transfected ex vivo to express NOD2.
30. The method of claim 29, wherein the cells are transfected using a cationic polymer, diethylaminoethyldextran, polyethylenimine, a cationic lipid, a liposome, calcium phosphate, an activated dendrimer, and/or a magnetic bead.
31. The method of claim 29 or 30, wherein the cells are transfected by way of electroporation, Nucleofection, squeeze-poration, sonoporation, optical transfection, Magnetofection, and/or impalefection.
32. The method of any one of claims 6-18, wherein the cells are obtained by delivering to the cells a nuclease that catalyzes a single-strand break or a double-strand break at a target position within the genome of the cell, optionally wherein the target position is near or within a gene encoding an endogenous NOD2 protein.
33. The method of claim 32, wherein the nuclease is delivered to the cells in combination with a guide RNA (gRNA) that hybridizes to the target position within the genome of the cell.
34. The method of claim 32 or 33, wherein the nuclease is a clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein.
35. The method of claim 34, wherein the CRISPR-associated protein is CRISPR-associated protein 9 (Cas9) or CRISPR-associated protein 12a (Cas12a).
36. The method of claim 32, wherein the nuclease is a transcription activator-like effector nuclease, a meganuclease, or a zinc finger nuclease.
37. The method of any one of claims 32-36, wherein the cells are additionally contacted with a template nucleic acid encoding NOD2 while the cells are contacted with the nuclease.
38. The method of claim 37, wherein the template nucleic acid molecule encoding NOD2 comprises a 5′ homology arm and a 3′ homology arm having nucleic acid sequences that are sufficiently similar to the nucleic acid sequences located 5′ to the target position and 3′ to the target position, respectively, to promote homologous recombination.
39. The method of claim 37 or 38, wherein the nuclease, gRNA, and template nucleic acid are delivered to the cells by contacting the cells with a viral vector that encodes the nuclease, gRNA, and template nucleic acid.
40. The method of claim 39, wherein the viral vector that encodes the nuclease, gRNA, and template nucleic acid is an adeno-associated virus (AAV), an adenovirus, a parvovirus, a coronavirus, a rhabdovirus, a paramyxovirus, a picornavirus, an alphavirus, a herpes virus, a poxvirus, or a Retroviridae family virus.
41. The method of claim 40, wherein the viral vector that encodes the nuclease, gRNA, and template nucleic acid is a Retroviridae family virus.
42. The method of claim 41, wherein the Retroviridae family virus is a lentiviral vector, alpharetroviral vector, or gammaretroviral vector.
43. The method of claim 41 or 42, wherein the Retroviridae family virus that encodes the nuclease, gRNA, and template nucleic acid comprises a central polypurine tract, a woodchuck hepatitis virus post-transcriptional regulatory element, a 5′-LTR, HIV signal sequence, HIV Psi signal 5′-splice site, delta-GAG element, 3′-splice site, and a 3′-self inactivating LTR.
44. The method of claim 43, wherein the viral vector that encodes the nuclease, gRNA, and template nucleic acid is an integration-deficient lentiviral vector (IDLV).
45. The method of claim 44, wherein the viral vector that encodes the nuclease, gRNA, and template nucleic acid is an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAVrh74, optionally wherein the AAV is AAV6.
46. The method of any one of claims 6-45, wherein prior to administering the composition to the patient, a population of precursor cells is isolated from the patient or a donor, and wherein the precursor cells are expanded ex vivo to yield the population of cells being administered to the patient.
47. The method of claim 46, wherein the precursor cells are CD34+ HSCs, and wherein the precursor cells are expanded without substantial loss of HSC functional potential.
48. The method of claim 46 or 47, wherein prior to isolation of the precursor cells from the patient or donor, the patient or donor is administered one or more pluripotent cell mobilization agents.
49. The method of any one of claims 6-48, wherein prior to administering the composition to the patient, a population of endogenous pluripotent cells is ablated in the patient by administration of one or more conditioning agents to the patient.
50. The method of any one of claims 6-48, the method comprising ablating a population of endogenous pluripotent cells in the patient by administering to the patient one or more conditioning agents prior to administering the composition to the patient.
51. The method of claim 49 or 50, wherein the one or more conditioning agents are non-myeloablative conditioning agents.
52. The method of any one of claims 6-51, wherein upon administration of the composition to the patient, the administered cells, or progeny thereof, differentiate into one or more cell types selected from megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeoblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T-lymphocytes, and B-lymphocytes.
53. The method of any one of claims 5-52, wherein the nucleic acid molecule comprises a transgene encoding NOD2 operably linked to a ubiquitous promoter, optionally wherein the promoter is an EF1α promoter or an EFS promoter.
54. The method of any one of claims 5-52, wherein the nucleic acid molecule comprises a transgene encoding NOD2 operably linked to a tissue-specific promoter.
55. The method of claim 54, wherein the promoter is a CD11b promoter, sp146/p47 promoter, CD68 promoter, sp146/gp9 promoter, or an endogenous NOD2 promoter.
56. The method of any one of claims 1-55, wherein the patient is a mammal.
57. The method of claim 56, wherein the patient is a human.
58. The method of any one of claims 1-57, wherein the patient has a loss-of-function mutation in an endogenous gene encoding NOD2.
59. The method of claim 58, wherein the mutation is selected from the group consisting of R702W, G908R, and L1007fs.
60. The method of claim 58 or 59, wherein the mutation is heterozygous.
61. The method of claim 58 or 59, wherein the mutation is homozygous.
62. The method of any one of claims 1-61, wherein the patient has previously been treated with one or more immunosuppressive agents, biologic agents, and/or corticosteroids.
63. The method of claim 62, wherein the patient has not responded to treatment with the one or more immunosuppressive agents, biologic agents, and/or corticosteroids.
64. The method of claim 62 or 63, wherein the one or more immunosuppressive agents comprise azathioprine, methotrexate and/or infliximab.
65. The method of any one of claims 1-64, wherein, prior to providing the patient with the one or more agents that increase expression and/or activity of NOD2, the patient exhibits persistent disease activity, as assessed by endoscopy, colonoscopy, and/or magnetic resonance enterography.
66. The method of any one of claims 1-65, wherein, prior to providing the patient with the one or more agents that increase expression and/or activity of NOD2, the patient has been determined to be at risk of short bowel disease and/or refractory colonic disease if the patient were to undergo an imminent surgical procedure.
67. The method of any one of claims 1-66, wherein, prior to providing the patient with the one or more agents that increase expression and/or activity of NOD2, the patient exhibits a persistent perianal lesion such that the patient is not a candidate for coloproctectomy.
68. The method of any one of claims 1-67, wherein, prior to providing the patient with the one or more agents that increase expression and/or activity of NOD2, the patient exhibits impaired function and/or quality of life.
69. The method of claim 68, wherein function and/or quality of life are assessed by way of an Inflammatory Bowel Disease Questionnaire (IBDQ), a European Questionnaire of Life Quality, or a Karnofsky Index.
70. The method of any one of claims 1-69, wherein, after providing the patient with the one or more agents that increase expression and/or activity of NOD2, the patient exhibits sustained disease remission, optionally wherein the patient exhibits the sustained disease remission one year after providing the patient with the one or more agents that increase expression and/or activity of NOD2.
71. The method of any one of claims 1-70, wherein, after providing the patient with the one or more agents that increase expression and/or activity of NOD2, the patient no longer requires treatment with immunosuppressive agents, biologic agents, and/or corticosteroids, optionally wherein the patient does not require treatment with the immunosuppressive agents, biologic agents, and/or corticosteroids for at least three months.
72. The method of any one of claims 1-71, wherein, after providing the patient with the one or more agents that increase expression and/or activity of NOD2, the patient does not exhibit evidence of erosive disease in the gastrointestinal tract, as assessed by endoscopy and/or radiology.
73. A pharmaceutical composition comprising (i) a population of cells that express NOD2, optionally wherein the cells are pluripotent cells, and (ii) one or more carriers, diluents, and/or excipients.
74. The pharmaceutical composition of claim 73, wherein the cells are human cells.
75. The pharmaceutical composition of claim 73 or 74, wherein the cells are HSCs or HPCs.
76. The pharmaceutical composition of claim 73 or 74, wherein the cells are embryonic stem cells.
77. The pharmaceutical composition of claim 73 or 74, wherein the cells are induced pluripotent stem cells.
78. The pharmaceutical composition of claim 73 or 74, wherein the cells are CD34+ cells.
79. The pharmaceutical composition of claim 78, wherein the CD34+ cells are myeloid progenitor cells.
80. The pharmaceutical composition of any one of claims 73-79, wherein the composition is formulated for administration to a human patient.
81. The pharmaceutical composition of claim 80, wherein the composition is formulated for intravenous injection to the human patient.
82. The pharmaceutical composition of claim 80 or 81, wherein the cells are autologous with respect to the patient.
83. The pharmaceutical composition of claim 80 or 81, wherein the cells are allogeneic with respect to the patient.
84. The pharmaceutical composition of claim 83, wherein the cells are HLA-matched to the patient.
85. The pharmaceutical composition of any one of claims 73-84, wherein the cells comprise a transgene encoding NOD2 operably linked to a ubiquitous promoter, optionally wherein the promoter is an EF1α promoter or an EFS promoter.
86. The pharmaceutical composition of any one of claims 73-84, wherein the cells comprise a transgene encoding NOD2 operably linked to a tissue-specific promoter.
87. The pharmaceutical composition of claim 86, wherein the promoter is a CD11b promoter, sp146/p47 promoter, CD68 promoter, sp146/gp9 promoter, or an endogenous NOD2 promoter.
88. A pharmaceutical composition comprising (i) a viral vector that encodes NOD2 and (ii) one or more carriers, diluents, and/or excipients.
89. The pharmaceutical composition of claim 88, wherein the viral vector is selected from the group consisting of a Retroviridae family virus, an adeno-associated virus, an adenovirus, a parvovirus, a coronavirus, a rhabdovirus, a paramyxovirus, a picornavirus, an alphavirus, a herpes virus, and a poxvirus.
90. The pharmaceutical composition of claim 89, wherein the viral vector is a Retroviridae family viral vector.
91. The pharmaceutical composition of claim 90, wherein the Retroviridae family viral vector is a lentiviral vector.
92. The pharmaceutical composition of claim 90, wherein the Retroviridae family viral vector is an alpharetroviral vector or a gammaretroviral vector.
93. The pharmaceutical composition of any one of claims 90-92, wherein the Retroviridae family viral vector comprises a central polypurine tract, a woodchuck hepatitis virus post-transcriptional regulatory element, a 5′-LTR, HIV signal sequence, HIV Psi signal 5′-splice site, delta-GAG element, 3′-splice site, and a 3′-self inactivating LTR.
94. The pharmaceutical composition of any one of claims 90-93, wherein the viral vector is a pseudotyped viral vector.
95. The pharmaceutical composition of claim 94, wherein the pseudotyped viral vector selected from the group consisting of a pseudotyped adenovirus, a pseudotyped parvovirus, a pseudotyped coronavirus, a pseudotyped rhabdovirus, a pseudotyped paramyxovirus, a pseudotyped picornavirus, a pseudotyped alphavirus, a pseudotyped herpes virus, a pseudotyped poxvirus, and a pseudotyped Retroviridae family virus.
96. The pharmaceutical composition of claim 94 or 95, wherein the pseudotyped viral vector comprises one or more envelope proteins from a virus selected from vesicular stomatitis virus (VSV), RD114 virus, murine leukemia virus (MLV), feline leukemia virus (FeLV), Venezuelan equine encephalitis virus (VEE), human foamy virus (HFV), walleye dermal sarcoma virus (WDSV), Semliki Forest virus (SFV), Rabies virus, avian leukosis virus (ALV), bovine immunodeficiency virus (BIV), bovine leukemia virus (BLV), Epstein-Barr virus (EBV), Caprine arthritis encephalitis virus (CAEV), Sin Nombre virus (SNV), Cherry Twisted Leaf virus (ChTLV), Simian T-cell leukemia virus (STLV), Mason-Pfizer monkey virus (MPMV), squirrel monkey retrovirus (SMRV), Rous-associated virus (RAV), Fujinami sarcoma virus (FuSV), avian carcinoma virus (MH2), avian encephalomyelitis virus (AEV), Alfa mosaic virus (AMV), avian sarcoma virus CT10, and equine infectious anemia virus (EIAV).
97. The pharmaceutical composition of claim 96, wherein the pseudotyped viral vector comprises a VSV-G envelope protein.
98. The pharmaceutical composition of any one of claims 88-97, wherein the composition is formulated for administration to a human patient.
99. The pharmaceutical composition of claim 98, wherein the composition is formulated for intravenous injection to the human patient.
100. The pharmaceutical composition of any one of claims 88-99, wherein the viral vector comprises a transgene encoding NOD2 operably linked to a ubiquitous promoter, optionally wherein the promoter is an EF1α promoter or an EFS promoter.
101. The pharmaceutical composition of any one of claims 88-100, wherein the viral vector comprises a transgene encoding NOD2 operably linked to a tissue-specific promoter.
102. The pharmaceutical composition of claim 101, wherein the promoter is a CD11b promoter, sp146/p47 promoter, CD68 promoter, sp146/gp9 promoter, or an endogenous NOD2 promoter.
103. A kit comprising the pharmaceutical composition of any one of claims 73-102, wherein the kit further comprises a package insert instructing a user of the kit to administer the pharmaceutical composition to a human patient having Crohn's disease.
104. The kit of claim 103, wherein the package insert instructs a user of the kit to perform the method of any one of claims 1-72.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0207]
[0208]
[0209]
[0210]
[0211]
[0212]
[0213]
[0214]
[0215]
[0216]
[0217]
[0218]
[0219]
[0220]
DETAILED DESCRIPTION
[0221] The present disclosure provides compositions and methods for treating or preventing Crohn's disease. The compositions and methods described herein may be used, for example, to treat a patient, such as an adult human patient, that is suffering from Crohn's disease, as well as to prophylactically treat a patient at risk of developing Crohn's disease. Patients may be treated, for example, by providing to the patients one or more agents that elevate the expression and/or activity of functional Nucleotide-binding oligomerization domain-containing protein 2 (NOD2), such as a population of cells (e.g., a population of pluripotent cells, such as hematopoietic stem cells) that express functional NOD2. Without being limited by mechanism, the provision of such agents may treat an underlying cause of the disease and reverse its pathophysiology. Thus, using the compositions and methods described herein, a patient may not only be treated in a manner that alleviates one or more symptoms associated with Crohn's disease, but also in a curative fashion or preventative fashion.
NOD2 Signal Transduction
[0222] NOD2 is an intracellular pattern recognition receptor (PRR), which recognizes bacterial pathogens and initiates an immune response accordingly. As a PRR, NOD2 recognizes bacterial lipopolysaccharide (LPS), muramyldipeptide (MDP), and other pathogen-associated molecular patterns (PAMPs). NOD2 is a 110 kDa cytoplasmic protein belonging to the Nod-like receptor (NLR) family. Its expression is largely restricted to monocytes and other antigen-presenting cells (APCs). Proteins in the NLR family generally contain a nucleotide-binding oligomerization domain (NOD) responsible for coordinating protein oligomerization. NOD2 also contains a C-terminal leucine-rich repeat (LRR) domain with 11 LRR repeats. The LRR domain coordinates ligand binding and other protein-protein interactions. NOD2 also contains two N-terminal caspase associated recruitment domains (CARDs). The CARDs recruit proteins to promote apoptosis. The NOD2 CARDs are also capable of activating NFκB signaling via a homophilic CARD-CARD interaction with RICK, a serine-threonine kinase. RICK then associates with IKKγ to promote IKK-dependent activation of NFκB.
[0223] Using the compositions and methods of the disclosure, an agent that increases NOD2 activity and/or expression, such as a cell (e.g., a CD34+ cell or other pluripotent cell described herein) that expresses NOD2, can be administered to a patient suffering from Crohn's disease (e.g., a patient having a defect in NOD2 expression) so as to promote the following beneficial characteristics:
[0224] (i) restoration of physiologically normal levels of NOD2 expression;
[0225] (ii) an increase in MDP detection;
[0226] (iii) an increase in NFκB signal transduction; and/or
[0227] (iv) an augmented immune response against pathogenic microbes.
[0228] Without being limited by mechanism, the section that follows describes how agents that increase the NOD2 activity and/or expression and effectuate one or more, or all, of the beneficial phenotypes described above can be used to treat Crohn's disease.
Crohn's Disease
Etiology and NOD2 Restoration Therapy
[0229] Crohn's disease is an autoinflammatory diseases that can be caused by defective NOD2 activity. This aberration in NOD2 activity can be triggered by mutations clustered in the nucleotide-binding oligomerization domain (NBD) of NOD2. Such mutations include R702W, G908R, L1007fs.
[0230] NOD2 is usually maintained in an inactive, autoinhibited conformation in the cell by way of interactions between the NACHT and leucine-rich repeat (LRR) domains, as well as interaction with cellular chaperones. NOD2 is activated upon recognition of MDP, through direct peptide interaction with the LRR. NOD2 and MDP association induces a conformational change based activation of the NOD2 protein. However, mutations in NOD2, such as those described above, prevent interaction and sensing of MDP, and subsequent activation of the NOD2 protein.
[0231] Using the compositions and methods of the disclosure, a patient, such as a human patient suffering from Crohn's disease, may be administered an agent that expresses a functional NOD2 protein that does not contain an activity-disrupting mutation. Exemplary agents that achieve this effect are pluripotent cells, such as hematopoietic stem cells and hematopoietic progenitor cells, that express functional NOD2. The sections that follow describe exemplary procedures for producing such agents, as well as how such agents may be used to treat a patient suffering from Crohn's disease.
Diagnosis
[0232] A patient (e.g., a human patient) can be diagnosed as having Crohn's disease in a variety of ways. Genetic testing offers one avenue by which a patient may be diagnosed as having (or at risk of developing) Crohn's disease. For example, a genetic analysis can be used to determine whether a patient has a loss-of-function mutation in an endogenous gene encoding NOD2, such as a mutation in a NOD2 gene selected from the group consisting of R702W, G908R, and L1007fs. Exemplary genetic tests that can be used to determine whether a patient has such a mutation include polymerase chain reaction (PCR) methods known in the art and described herein, among others.
[0233] Clinically, Crohn's disease may be detected, for example, by way of a blood test. In this setting, Crohn's disease may manifest as anemia, a condition characterized by an insufficiency of red blood cells to deliver oxygen to tissues. Crohn's disease may also manifest in the form of infection (e.g., a bacterial infection), which can be detected in blood by identifying bacterial nucleic acids, for example, using molecular biology techniques known in the art, such as PCR-based methods, among others.
[0234] Another indicator of Crohn's disease is the presence of occult blood in a patient's stool. This can readily be assessed upon analysis of a stool sample obtained from the patient.
[0235] Crohn's disease may also be detected by way of a colonoscopy. This procedure allows inspection of the entire colon and the end of the ileum. Clusters of inflammatory granulomas, if present, may confirm a diagnosis of Crohn's disease.
[0236] Another useful procedure for diagnosing a patient as having Crohn's disease is computerized tomography (CT). A CT scan may be used to inspect the entire bowel as well as at tissues exterior to the bowel. This technique allows the detection of complications associate with Crohn's disease, including abscesses, fistulas, and intestinal blockages.
[0237] A further procedure that can be used to facilitate a diagnosis of Crohn's disease is magnetic resonance imaging (MRI). MRI is particularly useful for evaluating a fistula proximal to the anus (e.g., detectable by way of a pelvic MRI) or the small intestine (e.g., detectable by way of MR enterography). Either or both may be indicative of Crohn's disease.
[0238] Another technique that may be used to detect Crohn's disease in a patient is capsule endoscopy. In this procedure, the patient swallows a capsule containing a microscale camera, which visualizes the small intestine. The images thus obtained may be inspected for signs of infection, which may be indicative of Crohn's disease.
[0239] In yet another technique, one may be diagnosed as having Crohn's disease by way of balloon-assisted enteroscopy. In this procedure, a scope is used to visualize further into the small bowel, particularly in regions not accessible to standard endoscopes. This technique is often useful when a capsule endoscopy reveals abnormalities, but the diagnosis is still in question.
Prevention
[0240] Using the compositions and methods described herein, a subject (e.g., a human subject) may be administered one or more agents that increase activity and/or expression of functional NOD2 (e.g., to within physiologically normal levels) so as to prevent the onset of Crohn's disease. The subject may be one that is at risk of developing Crohn's disease, but has not yet presented with an observable symptom of the disease. For example, the subject may be one that has a loss-of-function mutation in an endogenous gene encoding NOD2, such as a mutation in a NOD2 gene selected from the group consisting of R702W, G908R, and L1007fs. As described above, a subject can be identified as having such a mutation using standard molecular biology techniques known in the art and described herein, including PCR-based methodologies, among others.
Methods of Producing Functional NOD2-Expressing Cells by Viral Transduction
Transduction Using a Protein Kinase C Modulator
[0241] A variety of agents can be used to reduce PKC activity and/or expression. Without being limited by mechanism, such agents can augment viral transduction by stimulating Akt signal transduction and/or maintaining cofilin in a dephosphorylated state, thereby promoting actin depolymerization. This actin depolymerization event may serve to remove a physical barrier that hinders entry of a viral vector into the nucleus of a target cell.
[0242] Staurosporine and Variants Thereof
[0243] In some embodiments, the substance that reduces activity and/or expression of PKC is a PKC inhibitor. The PKC inhibitor may be staurosporine or a variant thereof. For example, the PKC inhibitor may be a compound represented by formula (I)
##STR00006##
[0244] wherein R.sub.1 is H, OH, optionally substituted alkoxy, optionally substituted acyloxy, optionally substituted amino, optionally substituted alkylamino, optionally substituted amido, halogen, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkenyl, optionally substituted C.sub.2-6 alkynyl, optionally substituted acyl, optionally substituted alkoxycarbonyl, oxo, thiocarbonyl, optionally substituted carboxy, or ureido;
[0245] R.sub.2 is H, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkenyl, optionally substituted C.sub.2-6 alkynyl, or optionally substituted acyl;
[0246] R.sub.a and R.sub.b are each, independently, H, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkenyl, or optionally substituted C.sub.2-6 alkynyl, optionally substituted and optionally fused aryl, optionally substituted and optionally fused heteroaryl, optionally substituted and optionally fused cycloalkyl, or optionally substituted and optionally fused heterocycloalkyl, or R.sub.a and R.sub.b, together with the atoms to which they are bound, are joined to form an optionally substituted and optionally fused heterocycloalkyl ring;
[0247] R.sub.c is O, NR.sub.d, or S;
[0248] R.sub.d is H, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkenyl, or optionally substituted C.sub.2-6 alkynyl;
[0249] each X is, independently, halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkyl sulfonyl, optionally substituted aryl sulfonyl, optionally substituted heteroaryl sulfonyl, optionally substituted cycloalkyl sulfonyl, optionally substituted heterocycloalkyl sulfonyl, optionally substituted alkyl sulfanyl, optionally substituted aryl sulfanyl, optionally substituted heteroaryl sulfanyl, optionally substituted cycloalkyl sulfanyl, optionally substituted heterocycloalkyl sulfanyl, optionally substituted alkyl sulfinyl, optionally substituted aryl sulfinyl, optionally substituted heteroaryl sulfinyl, optionally substituted cycloalkyl sulfinyl, optionally substituted heterocycloalkyl sulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and optionally fused aryl, optionally substituted and optionally fused heteroaryl, optionally substituted and optionally fused cycloalkyl, or optionally substituted and optionally fused heterocycloalkyl;
[0250] each Y is, independently, halogen, optionally substituted haloalkyl, cyano, optionally substituted amino, hydroxyl, thiol, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted acyloxy, optionally substituted alkoxycarbonyl, optionally substituted carboxy, ureido, optionally substituted alkyl sulfonyl, optionally substituted aryl sulfonyl, optionally substituted heteroaryl sulfonyl, optionally substituted cycloalkyl sulfonyl, optionally substituted heterocycloalkyl sulfonyl, optionally substituted alkyl sulfanyl, optionally substituted aryl sulfanyl, optionally substituted heteroaryl sulfanyl, optionally substituted cycloalkyl sulfanyl, optionally substituted heterocycloalkyl sulfanyl, optionally substituted alkyl sulfinyl, optionally substituted aryl sulfinyl, optionally substituted heteroaryl sulfinyl, optionally substituted cycloalkyl sulfinyl, optionally substituted heterocycloalkyl sulfinyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted and optionally fused aryl, optionally substituted and optionally fused heteroaryl, optionally substituted and optionally fused cycloalkyl, or optionally substituted and optionally fused heterocycloalkyl;
[0251] represents a bond that is optionally present;
[0252] n is an integer from 0-4; and
[0253] m is an integer from 0-4;
[0254] or a salt thereof.
[0255] Interfering RNA
[0256] Exemplary PKC modulating agents that may be used in conjunction with the compositions and methods of the disclosure include interfering RNA molecules, such as short interfering RNA (siRNA), short hairpin RNA (shRNA), and/or micro RNA (miRNA), that diminish PKC gene expression. Methods for producing interfering RNA molecules are known in the art and are described in detail, for example, in WO 2004/044136 and U.S. Pat. No. 9,150,605, the disclosures of each of which are incorporated herein by reference in their entirety.
Transduction Using an HDAC Inhibitor
[0257] A variety of agents can be used to inhibit histone deacetylases in order to increase the expression of a transgene during viral transduction. Without wishing to be bound by theory, reduced transgene expression from viral vectors may be caused by epigenetic silencing of vector genomes carried out by histone deacetylates. Hydroxamic acids represent a particularly robust class of HDAC inhibitors that inhibit these enzymes by virtue of hydroxamate functionality that binds cationic zinc within the active sites of these enzymes. Exemplary inhibitors include trichostatin A, as well as Vorinostat (N-hydroxy-N′-phenyl-octanediamide, described in Marks et al., Nature Biotechnology 25, 84 to 90 (2007); Stenger, Community Oncology 4, 384-386 (2007), the disclosures of which are incorporated by reference herein). Other HDAC inhibitors include Panobinostat, described in Drugs of the Future 32(4): 315-322 (2007), the disclosure of which is incorporated herein by reference.
[0258] Additional examples of hydroxamic acid inhibitors of histone deacetylases include the compounds shown below, described in Bertrand, European Journal of Medicinal Chemistry 45:2095-2116 (2010), the disclosure of which is incorporated herein by reference.
[0259] Other HDAC inhibitors that do not contain a hydroxamate substituent have also been developed, including Valproic acid (Gottlicher, et al., EMBO J. 20(24): 6969-6978 (2001) and Mocetinostat (N-(2-Aminophenyl)-4-[[(4-pyridin-3-ylpyrimidin-2-yl)amino]methyl]benzamide, described in Balasubramanian et al., Cancer Letters 280: 211-221 (2009)), the disclosure of each of which is incorporated herein by reference. Other small molecule inhibitors that exploit chemical functionality distinct from a hydroxamate include those described in Bertrand, European Journal of Medicinal Chemistry 45:2095-2116 (2010), the disclosure of which is incorporated herein by reference.
[0260] Additional examples of chemical modulators of histone acetylation useful with the compositions and methods of the invention include modulators of HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, Sirt1, Sirt2, and/or HAT, such as butyrylhydroxamic acid, M344, LAQ824 (Dacinostat), AR-42, Belinostat (PXD101), CUDC-101, Scriptaid, Sodium Phenylbutyrate, Tasquinimod, Quisinostat (JNJ-26481585), Pracinostat (SB939), CUDC-907, Entinostat (MS-275), Mocetinostat (MGCD0103), Tubastatin A HCl, PCI-34051, Droxinostat, PCI-24781 (Abexinostat), RGFP966, Rocilinostat (ACY-1215), CI994 (Tacedinaline), Tubacin, RG2833 (RGFP109), Resminostat, Tubastatin A, BRD73954, BG45, 4SC-202, CAY10603, LMK-235, Nexturastat A, TMP269, HPOB, Cambinol, and Anacardic Acid.
[0261] In some particular embodiments, the HDAC inhibitor is Scriptaid.
Transduction Using a Cyclosporine
[0262] In some embodiments, therapeutic cells of the disclosure are produced by transducing the cells in the presence of a cyclosporine, such as cyclosporine A (CsA) or cyclosporine H (CsH).
[0263] In some embodiments, the concentration of the cyclosporine, when contacted with the cell, is from about 1 μM to about 10 μM (e.g., about 1 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2 μM, 2.1 μM, 2.2 μM, 2.3 μM, 2.4 μM, 2.5 μM, 2.6 μM, 2.7 μM, 2.8 μM, 2.9 μM, 3 μM, 3.1 μM, 3.2 μM, 3.3 μM, 3.4 μM, 3.5 μM, 3.6 μM, 3.7 μM, 3.8 μM, 3.9 μM, 4 μM, 4.1 μM, 4.2 μM, 4.3 μM, 4.4 μM, 4.5 μM, 4.6 μM, 4.7 μM, 4.8 μM, 4.9 μM, 5 μM, 5.1 μM, 5.2 μM, 5.3 μM, 5.4 μM, 5.5 μM, 5.6 μM, 5.7 μM, 5.8 μM, 5.9 μM, 6 μM, 6.1 μM, 6.2 μM, 6.3 μM, 6.4 μM, 6.5 μM, 6.6 μM, 6.7 μM, 6.8 μM, 6.9 μM, 7 μM, 7.1 μM, 7.2 μM, 7.3 μM, 7.4 μM, 7.5 μM, 7.6 μM, 7.7 μM, 7.8 μM, 7.9 μM, 8 μM, 8.1 μM, 8.2 μM, 8.3 μM, 8.4 μM, 8.5 μM, 8.6 μM, 8.7 μM, 8.8 μM, 8.9 μM, 9 μM, 9.1 μM, 9.2 μM, 9.3 μM, 9.4 μM, 9.5 μM, 9.6 μM, 9.7 μM, 9.8 μM, 9.9 μM, or 10 μM).
Transduction Using an Activator of Prostaglandin E Receptor Signaling
[0264] In some embodiments, therapeutic cells of the disclosure are produced by transducing the cells in the presence of an activator of prostaglandin E receptor signaling.
[0265] In some embodiments, the activator of prostaglandin E receptor signaling is a small molecule, such as a compound described in WO 2007/112084 or WO 2010/108028, the disclosures of each of which are incorporated herein by reference as they pertain to prostaglandin E receptor signaling activators.
[0266] In some embodiments, the activator of prostaglandin E receptor signaling is a small molecule, such as a small organic molecule, a prostaglandin, a Wnt pathway agonist, a cAMP/PI3K/AKT pathway agonist, a Ca.sup.2+ second messenger pathway agonist, a nitric oxide (NO)/angiotensin signaling agonist, or another compound known to stimulate the prostaglandin signaling pathway, such as a compound selected from Mebeverine, Flurandrenolide, Atenolol, Pindolol, Gaboxadol, Kynurenic Acid, Hydralazine, Thiabendazole, Bicuclline, Vesamicol, Peruvoside, Imipramine, Chlorpropamide, 1,5-Pentamethylenetetrazole, 4-Aminopyridine, Diazoxide, Benfotiamine, 12-Methoxydodecenoic acid, N-Formyl-Met-Leu-Phe, Gallamine, IAA 94, Chlorotrianisene, and or a derivative of any of these compounds.
[0267] In some embodiments, the activator of prostaglandin E receptor signaling is a naturally-occurring or synthetic chemical molecule or polypeptide that binds to and/or interacts with a prostaglandin E receptor, typically to activate or increase one or more of the downstream signaling pathways associated with a prostaglandin E receptor.
[0268] In some embodiments, the activator of prostaglandin E receptor signaling is selected from the group consisting of prostaglandin (PG) A2 (PGA2), PGB2, PGD2, PGE1 (Alprostadil), PGE2, PGF2, PGI2 (Epoprostenol), PGH2, PGJ2, and derivatives and analogs thereof.
[0269] In some embodiments, the activator of prostaglandin E receptor signaling is PGE2 or dmPGE2.
[0270] In some embodiments, the activator of prostaglandin E receptor signaling is 15d-PGJ2, delta12-PGJ2, 2-hydroxyheptadecatrienoic acid (HHT), Thromboxane (TXA2 and TXB2), PGI2 analogs, e.g., Iloprost and Treprostinil, PGF2 analogs, e.g., Travoprost, Carboprost tromethamine, Tafluprost, Latanoprost, Bimatoprost, Unoprostone isopropyl, Cloprostenol, Oestrophan, and Superphan, PGE1 analogs, e.g., 11-deoxy PGE1, Misoprostol, and Butaprost, and Corey alcohol-A ([3aa,4a,5,6aa]-(−)-[Hexahydro-4-(hydroxymetyl)-2-oxo-2H-cyclopenta/b/furan-5-yl][1,1′-biphenyl]-4-carboxylate), Corey alcohol-B (2H-Cyclopenta[b]furan-2-on,5-(benzoyloxy)hexahydro-4-(hydroxymethyl)[3aR-(3aa,4a,5,6aa)]), and Corey diol ((3aR,4S,5R,6aS)-hexahydro-5-hydroxy-4-(hydroxymethyl)-2H-cyclopenta[b]furan-2-one).
[0271] In some embodiments, the activator of prostaglandin E receptor signaling is a prostaglandin E receptor ligand, such as prostaglandin E2 (PGE2), or an analogs or derivative thereof. Prostaglandins refer generally to hormone-like molecules that are derived from fatty acids containing 20 carbon atoms, including a 5-carbon ring, as described herein and known in the art. Illustrative examples of PGE2 “analogs” or “derivatives” include, but are not limited to, 16,16-dimethyl PGE2, 16-16 dimethyl PGE2 p-(p-acetamidobenzamido) phenyl ester, II-deoxy-16,16-dimethyl PGE2, 9-deoxy-9-methylene-16, 16-dimethyl PGE2, 9-deoxy-9-methylene PGE2, 9-keto Fluprostenol, 5-trans PGE2, 17-phenyl-omega-trinor PGE2, PGE2 serinol amide, PGE2 methyl ester, 16-phenyl tetranor PGE2, 15(S)-15-methyl PGE2, 15 (R)-15-methyl PGE2, 8-iso-15-keto PGE2, 8-iso PGE2 isopropyl ester, 20-hydroxy PGE2, nocloprost, sulprostone, butaprost, 15-keto PGE2, and 19 (R) hydroxy PGE2.
[0272] In some embodiments, the activator of prostaglandin E receptor signaling is a prostaglandin analog or derivative having a similar structure to PGE2 that is substituted with halogen at the 9-position (see, e.g., WO 2001/12596, herein incorporated by reference in its entirety), as well as 2-decarboxy-2-phosphinico prostaglandin derivatives, such as those described in US 2006/0247214, herein incorporated by reference in its entirety).
[0273] In some embodiments, the activator of prostaglandin E receptor signaling is a non-PGE2-based ligand. In some embodiments, the activator of prostaglandin E receptor signaling is CAY10399, ONO_8815Ly, ONO-AE1-259, or CP-533,536. Additional examples of non-PGE2-based EP2 agonists include the carbazoles and fluorenes disclosed in WO 2007/071456, herein incorporated by reference for its disclosure of such agents. Illustrative examples of non-PGE2-based EP3 agonist include, but are not limited to, AE5-599, MB28767, GR 63799X, ONO-NT012, and ONO-AE-248. Illustrative examples of non-PGE2-based EP4 agonist include, but are not limited to, ONO-4819, APS-999 Na, AH23848, and ONO-AE 1-329. Additional examples of non-PGE2-based EP4 agonists can be found in WO 2000/038663; U.S. Pat. Nos. 6,747,037; and 6,610,719, each of which are incorporated by reference for their disclosure of such agonists
[0274] In some embodiments, the activator of prostaglandin E receptor signaling is a Wnt agonist. Illustrative examples of Wnt agonists include, but are not limited to, Wnt polypeptides and glycogen synthase kinase 3 (GSK3) inhibitors. Illustrative examples of Wnt polypeptides suitable for use as compounds that stimulate the prostaglandin EP receptor signaling pathway include, but are not limited to, Wnt1, Wnt2, Wnt2b/13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnt1Oa, Wnt1Ob, Wnt11, Wnt14, Wnt15, or biologically active fragments thereof. GSK3 inhibitors suitable for use as agents that stimulate the prostaglandin EP receptor signaling pathway bind to and decrease the activity of GSK3a, or GSK3. Illustrative examples of GSK3 inhibitors include, but are not limited to, BIO (6-bromoindirubin-3′-oxime), LiCl, Li.sub.2CO.sub.3, or other GSK-3 inhibitors, as exemplified in U.S. Pat. Nos. 6,057,117 and 6,608,063, as well as US 2004/0092535 and US 2004/0209878, and ATP-competitive, selective GSK-3 inhibitors CHIR-911 and CHIR-837 (also referred to as CT-99021/CHIR-99021 and CT-98023/CHIR-98023, respectively) (Chiron Corporation (Emeryville, Calif.)). The structure of CHIR-99021 is
##STR00007##
[0275] or a salt thereof.
The structure of CHIR-98023 is
##STR00008##
[0276] or a salt thereof.
[0277] In some embodiments, method further includes contacting the cell with a GSK3 inhibitor.
[0278] In some embodiments, the GSK3 inhibitor is CHIR-99021 or CHIR-98023.
[0279] In some embodiments, the GSK3 inhibitor is Li.sub.2CO.sub.3.
[0280] In some embodiments, the activator of prostaglandin E receptor signaling is an agent that increases signaling through the cAMP/P13K/AKT second messenger pathway, such as an agent selected from the group consisting of dibutyryl cAMP (DBcAMP), phorbol ester, forskolin, sclareline, 8-bromo-cAMP, cholera toxin (CTx), aminophylline, 2,4 dinitrophenol (DNP), norepinephrine, epinephrine, isoproterenol, isobutylmethylxanthine (IBMX), caffeine, theophylline (dimethylxanthine), dopamine, rolipram, iloprost, pituitary adenylate cyclase activating polypeptide (PACAP), and vasoactive intestinal polypeptide (VIP), and derivatives of these agents.
[0281] In some embodiments, the activator of prostaglandin E receptor signaling is an agent that increases signaling through the Ca.sup.2+ second messenger pathway, such as an agent selected from the group consisting of Bapta-AM, Fendiline, Nicardipine, and derivatives of these agents.
[0282] In some embodiments, the activator of prostaglandin E receptor signaling is an agent that increases signaling through the NO/Angiotensin signaling, such as an agent selected from the group consisting of L-Arg, Sodium Nitroprusside, Sodium Vanadate, Bradykinin, and derivatives thereof.
Transduction Using a Polycationic Polymer
[0283] In some embodiments, therapeutic cells of the disclosure are produced by transducing the cells in the presence of a polycationic polymer. In some embodiments, the polycationic polymer is polybrene, protamine sulfate, polyethylenimine, or a polyethylene glycol/poly-L-lysine block copolymer.
[0284] In some embodiments, the polycationic polymer is protamine sulfate.
[0285] In some embodiments, the cell is further contacted with an expansion agent during the transduction procedure. The cell may be, for example, a hematopoietic stem cell and the expansion agent may be a hematopoietic stem cell expansion agent, such as a hematopoietic stem cell expansion agent known in the art or described herein.
Additional Transduction Enhancers
[0286] In some embodiments of the methods described herein, during the transduction procedure, the cell is further contacted with an agent that inhibits mTOR signaling. The agent that inhibits mTOR signaling may be, for example, rapamycin, among other suppressors of mTOR signaling.
[0287] Additional transduction enhancers that may be used in conjunction with the compositions and methods of the disclosure include, for example, tacrolimus and vectorfusin.
Spinoculation
[0288] In some embodiments of the disclosure, a cell targeted for transduction may be spun e.g., by centrifugation, while being cultured with a viral vector (e.g., in combination with one or more additional agents described herein). This “spinoculation” process may occur with a centripetal force of, e.g., from about 200×g to about 2,000×g. The centripetal force may be, e.g., from about 300×g to about 1,200×g (e.g., about 300×g, 400×g, 500×g, 600×g, 700×g, 800×g, 900×g, 1,000×g, 1,100×g, or 1,200×g, or more). In some embodiments, the cell is spun for from about 10 minutes to about 3 hours (e.g., about 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, 120 minutes, 125 minutes, 130 minutes, 135 minutes, 140 minutes, 145 minutes, 150 minutes, 155 minutes, 160 minutes, 165 minutes, 170 minutes, 175 minutes, 180 minutes, or more). In some embodiments, the cell is spun at room temperature, such as at a temperature of about 25° C.
[0289] Exemplary transduction protocols involving a spinoculation step are described, e.g., in Millington et al., PLoS One 4:e6461 (2009); Guo et al., Journal of Virology 85:9824-9833 (2011); O'Doherty et al., Journal of Virology 74:10074-10080 (2000); and Federico et al., Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools, Methods in Molecular Biology 1448, Chapter 4 (2016), the disclosures of each of which are incorporated herein by reference.
Viral Vectors for NOD2 Expression
[0290] Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous genes into a mammalian cell. Viral genomes are particularly useful vectors for gene delivery as the polynucleotides contained within such genomes are typically incorporated into the nuclear genome of a mammalian cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration. Examples of viral vectors are a retrovirus (e.g., Retroviridae family viral vector), adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus, coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, human papilloma virus, human foamy virus, and hepatitis virus, for example. Examples of retroviruses are: avian leukosis-sarcoma, avian C-type viruses, mammalian C-type, B-type viruses, D-type viruses, oncoretroviruses, HTLV-BLV group, lentivirus, alpharetrovirus, gammaretrovirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, Virology, Third Edition (Lippincott-Raven, Philadelphia, (1996))). Other examples are murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are described, for example, in McVey et al., (U.S. Pat. No. 5,801,030), the teachings of which are incorporated herein by reference.
Retroviral Vectors
[0291] The delivery vector used in the methods and compositions described herein may be a retroviral vector. One type of retroviral vector that may be used in the methods and compositions described herein is a lentiviral vector. Lentiviral vectors (LVs), a subset of retroviruses, transduce a wide range of dividing and non-dividing cell types with high efficiency, conferring stable, long-term expression of the transgene. An overview of optimization strategies for packaging and transducing LVs is provided in Delenda, The Journal of Gene Medicine 6: S125 (2004), the disclosure of which is incorporated herein by reference.
[0292] The use of lentivirus-based gene transfer techniques relies on the in vitro production of recombinant lentiviral particles carrying a highly deleted viral genome in which the transgene of interest is accommodated. In particular, the recombinant lentivirus are recovered through the in trans coexpression in a permissive cell line of (1) the packaging constructs, i.e., a vector expressing the Gag-Pol precursors together with Rev (alternatively expressed in trans); (2) a vector expressing an envelope receptor, generally of an heterologous nature; and (3) the transfer vector, consisting in the viral cDNA deprived of all open reading frames, but maintaining the sequences required for replication, encapsidation, and expression, in which the sequences to be expressed are inserted.
[0293] A LV used in the methods and compositions described herein may include one or more of a 5′-Long terminal repeat (LTR), HIV signal sequence, HIV Psi signal 5′-splice site (SD), delta-GAG element, Rev Responsive Element (RRE), 3′-splice site (SA), elongation factor (EF) 1-alpha promoter and 3′-self inactivating LTR (SIN-LTR). The lentiviral vector optionally includes a central polypurine tract (cPPT) and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), as described in U.S. Pat. No. 6,136,597, the disclosure of which is incorporated herein by reference as it pertains to WPRE. The lentiviral vector may further include a pHR′ backbone, which may include for example as provided below.
[0294] The Lentigen LV described in Lu et al., Journal of Gene Medicine 6:963 (2004) may be used to express the DNA molecules and/or transduce cells. A LV used in the methods and compositions described herein may a 5′-Long terminal repeat (LTR), HIV signal sequence, HIV Psi signal 5′-splice site (SD), delta-GAG element, Rev Responsive Element (RRE), 3′-splice site (SA), elongation factor (EF) 1-alpha promoter and 3′-self inactivating LTR (SIN-LTR). It will be readily apparent to one skilled in the art that optionally one or more of these regions is substituted with another region performing a similar function.
[0295] Enhancer elements can be used to increase expression of modified DNA molecules or increase the lentiviral integration efficiency. The LV used in the methods and compositions described herein may include a nef sequence. The LV used in the methods and compositions described herein may include a cPPT sequence which enhances vector integration. The cPPT acts as a second origin of the (+)-strand DNA synthesis and introduces a partial strand overlap in the middle of its native HIV genome. The introduction of the cPPT sequence in the transfer vector backbone strongly increased the nuclear transport and the total amount of genome integrated into the DNA of target cells. The LV used in the methods and compositions described herein may include a Woodchuck Posttranscriptional Regulatory Element (WPRE). The WPRE acts at the transcriptional level, by promoting nuclear export of transcripts and/or by increasing the efficiency of polyadenylation of the nascent transcript, thus increasing the total amount of mRNA in the cells. The addition of the WPRE to LV results in a substantial improvement in the level of transgene expression from several different promoters, both in vitro and in vivo. The LV used in the methods and compositions described herein may include both a cPPT sequence and WPRE sequence. The vector may also include an IRES sequence that permits the expression of multiple polypeptides from a single promoter.
[0296] In addition to IRES sequences, other elements which permit expression of multiple polypeptides are useful. The vector used in the methods and compositions described herein may include multiple promoters that permit expression more than one polypeptide. The vector used in the methods and compositions described herein may include a protein cleavage site that allows expression of more than one polypeptide. Examples of protein cleavage sites that allow expression of more than one polypeptide are described in Klump et al., Gene Ther.; 8:811 (2001), Osborn et al., Molecular Therapy 12:569 (2005), Szymczak and Vignali, Expert Opin Biol Ther. 5:627 (2005), and Szymczak et al., Nat Biotechnol. 22:589 (2004), the disclosures of which are incorporated herein by reference as they pertain to protein cleavage sites that allow expression of more than one polypeptide. It will be readily apparent to one skilled in the art that other elements that permit expression of multiple polypeptides identified in the future are useful and may be utilized in the vectors suitable for use with the compositions and methods described herein.
[0297] The vector used in the methods and compositions described herein may, be a clinical grade vector.
Methods of Producing Functional NOD2-Expressing Cells by Ex Vivo Transfection
[0298] One platform that can be used to achieve therapeutically effective intracellular concentrations of one or more proteins described herein in mammalian cells is via the stable expression of genes encoding these agents (e.g., by integration into the nuclear or mitochondrial genome of a mammalian cell). These genes are polynucleotides that encode the primary amino acid sequence of the corresponding protein. In order to introduce such exogenous genes into a mammalian cell, these genes can be incorporated into a vector. Vectors can be introduced into a cell by a variety of methods, including transformation, transfection, direct uptake, projectile bombardment, and by encapsulation of the vector in a liposome. Examples of suitable methods of transfecting or transforming cells are calcium phosphate precipitation, electroporation, microinjection, infection, lipofection, and direct uptake. Such methods are described in more detail, for example, in Green et al., Molecular Cloning: A Laboratory Manual, Fourth Edition (Cold Spring Harbor University Press, New York (2014)); and Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York (2015)), the disclosures of each of which are incorporated herein by reference.
[0299] Genes encoding therapeutic proteins of the disclosure can also be introduced into mammalian cells by targeting a vector containing a gene encoding such an agent to cell membrane phospholipids. For example, vectors can be targeted to the phospholipids on the extracellular surface of the cell membrane by linking the vector molecule to a VSV-G protein, a viral protein with affinity for all cell membrane phospholipids. Such, a construct can be produced using methods well known to those of skill in the field.
[0300] Recognition and binding of the polynucleotide encoding one or more therapeutic proteins of the disclosure by mammalian RNA polymerase is important for gene expression. As such, one may include sequence elements within the polynucleotide that exhibit a high affinity for transcription factors that recruit RNA polymerase and promote the assembly of the transcription complex at the transcription initiation site. Such sequence elements include, e.g., a mammalian promoter, the sequence of which can be recognized and bound by specific transcription initiation factors and ultimately RNA polymerase. Examples of mammalian promoters have been described in Smith et al., Mol. Sys. Biol., 3:73, online publication, the disclosure of which is incorporated herein by reference.
[0301] Once a polynucleotide encoding one or more therapeutic proteins has been incorporated into the nuclear DNA of a mammalian cell, transcription of this polynucleotide can be induced by methods known in the art. For example expression can be induced by exposing the mammalian cell to an external chemical reagent, such as an agent that modulates the binding of a transcription factor and/or RNA polymerase to the mammalian promoter and thus regulates gene expression. The chemical reagent can serve to facilitate the binding of RNA polymerase and/or transcription factors to the mammalian promoter, e.g., by removing a repressor protein that has bound the promoter. Alternatively, the chemical reagent can serve to enhance the affinity of the mammalian promoter for RNA polymerase and/or transcription factors such that the rate of transcription of the gene located downstream of the promoter is increased in the presence of the chemical reagent. Examples of chemical reagents that potentiate polynucleotide transcription by the above mechanisms are tetracycline and doxycycline. These reagents are commercially available (Life Technologies, Carlsbad, Calif.) and can be administered to a mammalian cell in order to promote gene expression according to established protocols.
[0302] Other DNA sequence elements that may be included in polynucleotides for use in the compositions and methods described herein are enhancer sequences. Enhancers represent another class of regulatory elements that induce a conformational change in the polynucleotide containing the gene of interest such that the DNA adopts a three-dimensional orientation that is favorable for binding of transcription factors and RNA polymerase at the transcription initiation site. Thus, polynucleotides for use in the compositions and methods described herein include those that encode one or more therapeutic proteins and additionally include a mammalian enhancer sequence. Many enhancer sequences are now known from mammalian genes, and examples are enhancers from the genes that encode mammalian globin, elastase, albumin, α-fetoprotein, and insulin. Enhancers for use in the compositions and methods described herein also include those that are derived from the genetic material of a virus capable of infecting a eukaryotic cell. Examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Additional enhancer sequences that induce activation of eukaryotic gene transcription are disclosed in Yaniv et al., Nature 297:17 (1982).
Cells for Expression and Delivery of NOD2
[0303] Cells that may be used in conjunction with the compositions and methods described herein include cells that are capable of undergoing further differentiation. For example, one type of cell that can be used in conjunction with the compositions and methods described herein is a pluripotent cell. A pluripotent cell is a cell that possesses the ability to develop into more than one differentiated cell type. Examples of pluripotent cells are ESCs, iPSCs, and CD34+ cells. ESCs and iPSCs have the ability to differentiate into cells of the ectoderm, which gives rise to the skin and nervous system, endoderm, which forms the gastrointestinal and respiratory tracts, endocrine glands, liver, and pancreas, and mesoderm, which forms bone, cartilage, muscles, connective tissue, and most of the circulatory system.
[0304] Cells that may be used in conjunction with the compositions and methods described herein include hematopoietic stem cells and hematopoietic progenitor cells. Hematopoietic stem cells (HSCs) are immature blood cells that have the capacity to self-renew and to differentiate into mature blood cells including diverse lineages including but not limited to granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B-cells and T-cells). Human HSCs are CD34+. In addition, HSCs also refer to long term repopulating HSC (LT-HSC) and short-term repopulating HSC (ST-HSC). Any of these HSCs can be used in conjunction with the compositions and methods described herein.
[0305] HSCs and other pluripotent progenitors can be obtained from blood products. A blood product is a product obtained from the body or an organ of the body containing cells of hematopoietic origin. Such sources include unfractionated bone marrow, umbilical cord, placenta, peripheral blood, or mobilized peripheral blood. All of the aforementioned crude or unfractionated blood products can be enriched for cells having HSC or myeloid progenitor cell characteristics in a number of ways. For example, the more mature, differentiated cells can be selected against based on cell surface molecules they express. The blood product may be fractionated by positively selecting for CD34+ cells, which include a subpopulation of hematopoietic stem cells capable of self-renewal, multi-potency, and that can be re-introduced into a transplant recipient whereupon they home to the hematopoietic stem cell niche and reestablish productive and sustained hematopoiesis. Such selection is accomplished using, for example, commercially available magnetic anti-CD34 beads (Dynal, Lake Success, N.Y.). Myeloid progenitor cells can also be isolated based on the markers they express. Unfractionated blood products can be obtained directly from a donor or retrieved from cryopreservative storage. HSCs and myeloid progenitor cells can also be obtained from by differentiation of ES cells, iPS cells or other reprogrammed mature cells types.
[0306] Cells that may be used in conjunction with the compositions and methods described herein include allogeneic cells and autologous cells. When allogeneic cells are used, the cells may optionally be HLA-matched to the subject receiving a cell treatment.
[0307] Cells that may be used in conjunction with the compositions and methods described herein include CD34+/CD90+ cells and CD34+/CD164+ cells. These cells may contain a higher percentage of HSCs. These cells are described in Radtke et al. Sci. Transl. Med. 9: 1-10, 2017, and Pellin et al. Nat. Comm. 1-: 2395, 2019, the disclosures of each of which are hereby incorporated by reference in their entirety.
[0308] The cells described herein and above may be genetically modified so as to express NOD2 using, for example, a variety of methodologies (see, for example, the sections entitled “Methods of Producing Functional NOD2-Expressing Cells by Viral Transduction,” “Methods of Producing Functional NOD2-Expressing Cells by Ex Vivo Transfection,” and “Promoting Functional NOD2 Expression Using Gene Editing Techniques”). Once the cells have been adapted to express physiological levels of functional NOD2, these cells have therapeutic utility, and are referred to herein as “therapeutic cells of the disclosure.”
Promoting Functional NOD2 Expression Using Gene Editing Techniques
[0309] Another useful tool for the disruption and/or integration of target genes into the genome of a cell (e.g., a pluripotent stem cell) is the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system, a system that originally evolved as an adaptive defense mechanism in bacteria and archaea against viral infection. The CRISPR/Cas system includes palindromic repeat sequences within plasmid DNA and a CRISPR-associated protein (Cas; e.g., Cas9 or Cas12a). This ensemble of DNA and protein directs site specific DNA cleavage of a target sequence by first incorporating foreign DNA into CRISPR loci. Polynucleotides containing these foreign sequences and the repeat-spacer elements of the CRISPR locus are in turn transcribed in a host cell to create a guide RNA, which can subsequently anneal to a target sequence and localize the Cas nuclease to this site. In this manner, highly site-specific Cas-mediated DNA cleavage can be engendered in a foreign polynucleotide because the interaction that brings Cas within close proximity of the target DNA molecule is governed by RNA:DNA hybridization. As a result, one can design a CRISPR/Cas system to cleave any target DNA molecule of interest. This technique has been exploited in order to edit eukaryotic genomes (Hwang et al. Nature Biotechnology 31:227 (2013), the disclosure of which is incorporated herein by reference) and can be used as an efficient means of site-specifically editing pluripotent stem cell genomes in order to cleave DNA prior to the incorporation of a gene encoding a target gene. The use of CRISPR/Cas to modulate gene expression has been described in, e.g., WO 2017/182881 and U.S. Pat. No. 8,697,359, the disclosures of each of which are incorporated herein by reference.
[0310] For example, using the compositions and methods of the disclosure, a genetic locus containing a nucleic acid that encodes a defective NOD2 protein may be edited so as to recapitulate functional NOD2 expression. An exemplary procedure for doing so is shown in
[0311] Alternatively, base editing may be used to site-specifically edit one or more nucleobase at a desired site in the target cell genome so as to negate a NOD2 defect-causing mutation and recapitulate expression of a gene encoding functional NOD2. Base editing techniques may use, for example, a mutant Cas9 that induces a single-strand break in one strand of endogenous DNA in the target cell, at which point a fused deaminase then converts one base to another, such as adenine (A) to inosine (I), a proxy for guanine (G) following DNA replication. The accompanying T to C change in the remaining DNA strand occurs by way of DNA repair and replication. Base editing may also be used at the level of RNA, as mutant Cas13-ADAR fusion proteins have been deployed to bind RNA and catalyzing nucleobase modifications resulting in a change of A to I. Exemplary methods for DNA base editing that may be used to negate a defect-causing NOD2 mutation in the cells and recapitulate expression of a functional NOD2 protein are described in Cohen, “Novel CRISPR-derived ‘base editors’ surgically alter DNA or RNA, offering new ways to fix mutations,’ Science Magazine, October 2017, the disclosure of which is incorporated herein by reference.
[0312] Alternative methods for disruption of a target DNA by site-specifically cleaving genomic DNA prior to the incorporation of a gene of interest in a pluripotent stem cell include the use of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Unlike the CRISPR/Cas system, these enzymes do not contain a guiding polynucleotide to localize to a specific target sequence. Target specificity is instead controlled by DNA binding domains within these enzymes. The use of ZFNs and TALENs in genome editing applications is described, e.g., in Urnov et al. Nature Reviews Genetics 11:636 (201 O); and in Joung et al. Nature Reviews Molecular Cell Biology 14:49 (2013), the disclosures of each of which are incorporated herein by reference. In some embodiments, an endogenous gene is disrupted, e.g., in a pluripotent stem cell, using the gene editing techniques described above.
[0313] In some embodiments, a gene editing approach, such as a CRISPR/Cas system or another of the nucleases described above, is used in order to insert a gene encoding a functional NOD2 protein (i.e., a NOD2 protein lacking an activity-disrupting mutation) directly into an endogenous NOD2 locus in a cell obtained from a patient suffering from Crohn's disease. In this way, expression of mutant NOD2 may be suppressed while simultaneously inducing expression of a functional NOD2 protein.
Agents that Enhance Cellular Engraftment
[0314] In some embodiments, the one or more agents administered to a patient that increase activity or expression of functional NOD2 is a population of cells (e.g., CD34+ cells) that express a NOD2 transgene. In such instances, prior to administration of the cells to the patient, the patient may be administered an agent that ablates an endogenous population of CD34+ cells, allowing the administered CD34+ cells to engraft in the patient. Examples of conditioning agents include myeloablative conditioning agents, which deplete a wide variety of hematopoietic cells in a patient. For instance, that patient may be pre-treated with an alkylating agent, such as a nitrogen mustard (e.g., bendamustine, chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, or melphalan), a nitrosourea (e.g., carmustine, lomustine, or streptozocin), an alkyl sulfonate (e.g., busulfan), a triazine (e.g., dacarbazine or temozolomide), or an ethylenimine (e.g., altretamine or thiotepa). In some embodiments, the patient is administered a conditioning agent that selectively ablates a specific population of endogenous cells, such as a population of endogenous CD34+ HSCs or HPCs.
[0315] In some embodiments, the patient is pre-treated with an activator of prostaglandin E receptor signaling in order to help facilitate the engraftment of administered NOD2-expressing cells. The prostaglandin E receptor signaling activator may be, for example, selected from the group consisting of prostaglandin (PG) A2 (PGA2), PGB2, PGD2, PGE1 (Alprostadil), PGE2, PGF2, PGI2 (Epoprostenol), PGH2, PGJ2, and derivatives and analogs thereof.
[0316] In some embodiments, the activator of prostaglandin E receptor signaling used to help facilitate engraftment of a NOD2-expressing cell is PGE2 or dmPG2.
[0317] In some embodiments, the activator of prostaglandin E receptor signaling used to help facilitate engraftment of a NOD2-expressing cell is 15d-PGJ2, delta12-PGJ2, 2-hydroxyheptadecatrienoic acid (HHT), Thromboxane (TXA2 and TXB2), PGI2 analogs, e.g., Iloprost and Treprostinil, PGF2 analogs, e.g., Travoprost, Carboprost tromethamine, Tafluprost, Latanoprost, Bimatoprost, Unoprostone isopropyl, Cloprostenol, Oestrophan, and Superphan, PGE1 analogs, e.g., 11-deoxy PGE1, Misoprostol, and Butaprost, and Corey alcohol-A ([3aa,4a,5,6aa]-(+[Hexahydro-4-(hydroxymetyl)-2-oxo-2H-cyclopenta/b/furan-5-yl][1,1′-biphenyl]-4-carboxylate), Corey alcohol-B (2H-Cyclopenta[b]furan-2-on,5-(benzoyloxy)hexahydro-4-(hydroxymethyl)[3aR-(3aa,4a,5,6aa)]), and Corey diol ((3aR,4S,5R,6aS)-hexahydro-5-hydroxy-4-(hydroxymethyl)-2H-cyclopenta[b]furan-2-one).
[0318] In some embodiments, the activator of prostaglandin E receptor signaling used to help facilitate engraftment of a NOD2-expressing cell is a prostaglandin E receptor ligand, such as prostaglandin E2 (PGE2), or an analogs or derivative thereof. Prostaglandins refer generally to hormone-like molecules that are derived from fatty acids containing 20 carbon atoms, including a 5-carbon ring, as described herein and known in the art. Illustrative examples of PGE2 “analogs” or “derivatives” include, but are not limited to, 16,16-dimethyl PGE2, 16-16 dimethyl PGE2 p-(p-acetamidobenzamido) phenyl ester, I I-deoxy-16,16-dimethyl PGE2, 9-deoxy-9-methylene-16, 16-dimethyl PGE2, 9-deoxy-9-methylene PGE2, 9-keto Fluprostenol, 5-trans PGE2, 17-phenyl-omega-trinor PGE2, PGE2 serinol amide, PGE2 methyl ester, 16-phenyl tetranor PGE2, 15(S)-15-methyl PGE2, 15 (R)-15-methyl PGE2, 8-iso-15-keto PGE2, 8-iso PGE2 isopropyl ester, 20-hydroxy PGE2, nocloprost, sulprostone, butaprost, 15-keto PGE2, and 19 (R) hydroxy PGE2.
[0319] In some embodiments, the activator of prostaglandin E receptor signaling used to help facilitate engraftment of a NOD2-expressing cell is a prostaglandin analog or derivative having a similar structure to PGE2 that is substituted with halogen at the 9-position (see, e.g., WO 2001/12596, herein incorporated by reference in its entirety), as well as 2-decarboxy-2-phosphinico prostaglandin derivatives, such as those described in US 2006/0247214, herein incorporated by reference in its entirety).
[0320] In some embodiments, the activator of prostaglandin E receptor signaling used to help facilitate engraftment of a NOD2-expressing cell is a non-PGE2-based ligand. In some embodiments, the activator of prostaglandin E receptor signaling used to help facilitate engraftment of a NOD2-expressing cell is CAY10399, ONO_8815Ly, ONO-AE1-259, or CP-533,536. Additional examples of non-PGE2-based EP2 agonists include the carbazoles and fluorenes disclosed in WO 2007/071456, herein incorporated by reference for its disclosure of such agents. Illustrative examples of non-PGE2-based EP.sub.3 agonist include, but are not limited to, AE5-599, MB28767, GR 63799X, ONO-NT012, and ONO-AE-248. Illustrative examples of non-PGE.sub.2-based EP.sub.4 agonist include, but are not limited to, ONO-4819, APS-999 Na, AH23848, and ONO-AE 1-329. Additional examples of non-PGE2-based EP4 agonists can be found in WO 2000/038663; U.S. Pat. Nos. 6,747,037; and 6,610,719, each of which are incorporated by reference for their disclosure of such agonists
[0321] In some embodiments, the activator of prostaglandin E receptor signaling used to help facilitate engraftment of a NOD2-expressing cell is a Wnt agonist. Illustrative examples of Wnt agonists include, but are not limited to, Wnt polypeptides and glycogen synthase kinase 3 (GSK3) inhibitors. Illustrative examples of Wnt polypeptides suitable for use as compounds that stimulate the prostaglandin EP receptor signaling pathway include, but are not limited to, Wnt1, Wnt2, Wnt2b/13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnt1Oa, Wnt1Ob, Wnt11, Wnt14, Wnt15, or biologically active fragments thereof. GSK3 inhibitors suitable for use as agents that stimulate the prostaglandin EP receptor signaling pathway bind to and decrease the activity of GSK3a, or GSK3. Illustrative examples of GSK3 inhibitors include, but are not limited to, BIO (6-bromoindirubin-3′-oxime), LiCl, Li.sub.2CO.sub.3 or other GSK-3 inhibitors, as exemplified in U.S. Pat. Nos. 6,057,117 and 6,608,063, as well as US 2004/0092535 and US 2004/0209878, and ATP-competitive, selective GSK-3 inhibitors CHIR-911 and CHIR-837 (also referred to as CT-99021/CHIR-99021 and CT-98023/CHIR-98023, respectively) (Chiron Corporation (Emeryville, Calif.)).
[0322] In some embodiments, the activator of prostaglandin E receptor signaling used to help facilitate engraftment of a NOD2-expressing cell is an agent that increases signaling through the cAMP/P13K/AKT second messenger pathway, such as an agent selected from the group consisting of dibutyryl cAMP (DBcAMP), phorbol ester, forskolin, sclareline, 8-bromo-cAMP, cholera toxin (CTx), aminophylline, 2,4 dinitrophenol (DNP), norepinephrine, epinephrine, isoproterenol, isobutylmethylxanthine (IBMX), caffeine, theophylline (dimethylxanthine), dopamine, rolipram, iloprost, pituitary adenylate cyclase activating polypeptide (PACAP), and vasoactive intestinal polypeptide (VIP), and derivatives of these agents.
[0323] In some embodiments, the activator of prostaglandin E receptor signaling used to help facilitate engraftment of a NOD2-expressing cell is an agent that increases signaling through the Ca.sup.2+ second messenger pathway, such as an agent selected from the group consisting of Bapta-AM, Fendiline, Nicardipine, and derivatives of these agents.
[0324] In some embodiments, the activator of prostaglandin E receptor signaling used to help facilitate engraftment of a NOD2-expressing cell is an agent that increases signaling through the NO/Angiotensin signaling, such as an agent selected from the group consisting of L-Arg, Sodium Nitroprusside, Sodium Vanadate, Bradykinin, and derivatives thereof.
Methods of Measuring NOD2 Gene Expression
[0325] Preferably, the compositions and methods of the disclosure are used to facilitate expression of functional NOD2 at physiologically normal levels in a patient (e.g., a human patient having Crohn's disease). The therapeutic agents of the disclosure, for example, may stimulate functional NOD2 expression in a human patient (e.g., a human patient suffering from Crohn's disease) that has a NOD2 deficiency. For example, the therapeutic agents of the disclosure may facilitate NOD2 expression in a Crohn's disease patient at a level of, for example, from about 20% to about 200% of the level of functional NOD2 expression observed in a human subject of comparable age and body mass index that does not have a NOD2 deficiency (e.g., about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or 200% of the level of functional NOD2 expression observed in a human subject of comparable age and body mass index that does not have a NOD2 deficiency).
[0326] The expression level of functional NOD2 expressed in a patient can be ascertained, for example, by evaluating the concentration or relative abundance of mRNA transcripts derived from transcription of a functional NOD2 transgene. Additionally or alternatively, gene expression can be determined by evaluating the concentration or relative abundance of functional NOD2 protein produced by transcription and translation of a NOD2 transgene. Protein concentrations can also be assessed using functional assays, such as MDP detection assays. The sections that follow describe exemplary techniques that can be used to measure the expression level of a NOD2 transgene upon delivery to a patient, such as a patient having Crohn's disease as described herein. Transgene expression can be evaluated by a number of methodologies known in the art, including, but not limited to, nucleic acid sequencing, microarray analysis, proteomics, in-situ hybridization (e.g., fluorescence in-situ hybridization (FISH)), amplification-based assays, in situ hybridization, fluorescence activated cell sorting (FACS), northern analysis and/or PCR analysis of mRNAs.
Nucleic Acid Detection
[0327] Nucleic acid-based methods for determining NOD2 transgene expression detection that may be used in conjunction with the compositions and methods described herein include imaging-based techniques (e.g., Northern blotting or Southern blotting). Such techniques may be performed using cells obtained from a patient following administration of the NOD2 transgene. Northern blot analysis is a conventional technique well known in the art and is described, for example, in Molecular Cloning, a Laboratory Manual, second edition, 1989, Sambrook, Fritch, Maniatis, Cold Spring Harbor Press, 10 Skyline Drive, Plainview, N.Y. 11803-2500. Typical protocols for evaluating the status of genes and gene products are found, for example in Ausubel et al., eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis).
[0328] Transgene detection techniques that may be used in conjunction with the compositions and methods described herein to evaluate NOD2 expression further include microarray sequencing experiments (e.g., Sanger sequencing and next-generation sequencing methods, also known as high-throughput sequencing or deep sequencing). Exemplary next generation sequencing technologies include, without limitation, Illumina sequencing, Ion Torrent sequencing, 454 sequencing, SOLiD sequencing, and nanopore sequencing platforms. Additional methods of sequencing known in the art can also be used. For instance, transgene expression at the mRNA level may be determined using RNA-Seq (e.g., as described in Mortazavi et al., Nat. Methods 5:621-628 (2008) the disclosure of which is incorporated herein by reference in their entirety). RNA-Seq is a robust technology for monitoring expression by direct sequencing the RNA molecules in a sample. Briefly, this methodology may involve fragmentation of RNA to an average length of 200 nucleotides, conversion to cDNA by random priming, and synthesis of double-stranded cDNA (e.g., using the Just cDNA DoubleStranded cDNA Synthesis Kit from Agilent Technology). Then, the cDNA is converted into a molecular library for sequencing by addition of sequence adapters for each library (e.g., from Illumina®/Solexa), and the resulting 50-100 nucleotide reads are mapped onto the genome.
[0329] Transgene expression levels may be determined using microarray-based platforms (e.g., single-nucleotide polymorphism arrays), as microarray technology offers high resolution. Details of various microarray methods can be found in the literature. See, for example, U.S. Pat. No. 6,232,068 and Pollack et al., Nat. Genet. 23:41-46 (1999), the disclosures of each of which are incorporated herein by reference in their entirety. Using nucleic acid microarrays, mRNA samples are reverse transcribed and labeled to generate cDNA. The probes can then hybridize to one or more complementary nucleic acids arrayed and immobilized on a solid support. The array can be configured, for example, such that the sequence and position of each member of the array is known. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene. Expression level may be quantified according to the amount of signal detected from hybridized probe-sample complexes. A typical microarray experiment involves the following steps: 1) preparation of fluorescently labeled target from RNA isolated from the sample, 2) hybridization of the labeled target to the microarray, 3) washing, staining, and scanning of the array, 4) analysis of the scanned image and 5) generation of gene expression profiles. One example of a microarray processor is the Affymetrix GENECHIP® system, which is commercially available and comprises arrays fabricated by direct synthesis of oligonucleotides on a glass surface. Other systems may be used as known to one skilled in the art.
[0330] Amplification-based assays also can be used to measure the expression level of a transgene in a target cell following delivery to a patient. In such assays, the nucleic acid sequences of the gene act as a template in an amplification reaction (for example, PCR, such as qPCR). In a quantitative amplification, the amount of amplification product is proportional to the amount of template in the original sample. Comparison to appropriate controls provides a measure of the expression level of the gene, corresponding to the specific probe used, according to the principles described herein. Methods of real-time qPCR using TaqMan probes are well known in the art. Detailed protocols for real-time qPCR are provided, for example, in Gibson et al., Genome Res. 6:995-1001 (1996), and in Heid et al., Genome Res. 6:986-994 (1996), the disclosures of each of which are incorporated herein by reference in their entirety. Levels of gene expression as described herein can be determined by RT-PCR technology. Probes used for PCR may be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator, or enzyme.
Protein Detection
[0331] Transgene expression can additionally be determined by measuring the concentration or relative abundance of a corresponding protein product (e.g., NOD2) encoded by a gene of interest. Protein levels can be assessed using standard detection techniques known in the art. Protein expression assays suitable for use with the compositions and methods described herein include proteomics approaches, immunohistochemical and/or western blot analysis, immunoprecipitation, molecular binding assays, ELISA, enzyme-linked immunofiltration assay (ELIFA), mass spectrometry, mass spectrometric immunoassay, and biochemical enzymatic activity assays. In particular, proteomics methods can be used to generate large-scale protein expression datasets in multiplex. Proteomics methods may utilize mass spectrometry to detect and quantify polypeptides (e.g., proteins) and/or peptide microarrays utilizing capture reagents (e.g., antibodies) specific to a panel of target proteins to identify and measure expression levels of proteins expressed in a sample (e.g., a single cell sample or a multi-cell population).
[0332] Exemplary peptide microarrays have a substrate-bound plurality of polypeptides, the binding of an oligonucleotide, a peptide, or a protein to each of the plurality of bound polypeptides being separately detectable. Alternatively, the peptide microarray may include a plurality of binders, including, but not limited to, monoclonal antibodies, polyclonal antibodies, phage display binders, yeast two-hybrid binders, aptamers, which can specifically detect the binding of specific oligonucleotides, peptides, or proteins. Examples of peptide arrays may be found in U.S. Pat. Nos. 6,268,210, 5,766,960, and 5,143,854, the disclosures of each of which are incorporated herein by reference in their entirety.
[0333] Mass spectrometry (MS) may be used in conjunction with the methods described herein to identify and characterize transgene expression in a cell from a patient (e.g., a human patient) following delivery of the transgene. Any method of MS known in the art may be used to determine, detect, and/or measure a protein or peptide fragment of interest, e.g., LC-MS, ESI-MS, ESI-MS/MS, MALDI-TOF-MS, MALDI-TOF/TOF-MS, tandem MS, and the like. Mass spectrometers generally contain an ion source and optics, mass analyzer, and data processing electronics. Mass analyzers include scanning and ion-beam mass spectrometers, such as time-of-flight (TOF) and quadruple (Q), and trapping mass spectrometers, such as ion trap (IT), Orbitrap, and Fourier transform ion cyclotron resonance (FT-ICR), may be used in the methods described herein. Details of various MS methods can be found in the literature. See, for example, Yates et al., Annu. Rev. Biomed. Eng. 11:49-79, 2009, the disclosure of which is incorporated herein by reference in its entirety.
[0334] Prior to MS analysis, proteins in a sample obtained from the patient can be first digested into smaller peptides by chemical (e.g., via cyanogen bromide cleavage) or enzymatic (e.g., trypsin) digestion. Complex peptide samples also benefit from the use of front-end separation techniques, e.g., 2D-PAGE, HPLC, RPLC, and affinity chromatography. The digested, and optionally separated, sample is then ionized using an ion source to create charged molecules for further analysis. Ionization of the sample may be performed, e.g., by electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), photoionization, electron ionization, fast atom bombardment (FAB)/liquid secondary ionization (LSIMS), matrix assisted laser desorption/ionization (MALDI), field ionization, field desorption, thermospray/plasmaspray ionization, and particle beam ionization. Additional information relating to the choice of ionization method is known to those of skill in the art.
[0335] After ionization, digested peptides may then be fragmented to generate signature MS/MS spectra. Tandem MS, also known as MS/MS, may be particularly useful for analyzing complex mixtures. Tandem MS involves multiple steps of MS selection, with some form of ion fragmentation occurring in between the stages, which may be accomplished with individual mass spectrometer elements separated in space or using a single mass spectrometer with the MS steps separated in time. In spatially separated tandem MS, the elements are physically separated and distinct, with a physical connection between the elements to maintain high vacuum. In temporally separated tandem MS, separation is accomplished with ions trapped in the same place, with multiple separation steps taking place over time. Signature MS/MS spectra may then be compared against a peptide sequence database (e.g., SEQUEST). Post-translational modifications to peptides may also be determined, for example, by searching spectra against a database while allowing for specific peptide modifications.
Routes of Administration
[0336] The compositions described herein may be administered to a patient (e.g., a human patient suffering from Crohn's disease) by one or more of a variety of routes, such as intravenously or by means of a bone marrow transplant. The most suitable route for administration in any given case may depend on the particular composition administered, the patient, pharmaceutical formulation methods, administration methods (e.g., administration time and administration route), the patients age, body weight, sex, severity of the diseases being treated, the patient's diet, and the patient's excretion rate. Multiple routes of administration may be used to treat a single patient at one time, or the patient may receive treatment via one route of administration first, and receive treatment via another route of administration during a second appointment, e.g., 1 week later, 2 weeks later, 1 month later, 6 months later, or 1 year later. Compositions may be administered to a subject once, or cells may be administered one or more times (e.g., 2-10 times) per week, month, or year.
Selection of Donor Cells
[0337] In some embodiments, the patient undergoing treatment is the donor that provides cells (e.g., pluripotent cells, such as CD34+ hematopoietic stem or progenitor cells) that are subsequently modified to express one or more therapeutic proteins of the disclosure before being re-administered to the patient. In such cases, withdrawn cells (e.g., hematopoietic stem or progenitor cells) may be re-infused into the subject following, for example, incorporation of a transgene encoding functional NOD2, such that the cells may subsequently home to hematopoietic tissue and establish productive hematopoiesis, thereby populating or repopulating a line of cells that is defective or deficient in the patient. In cases in which the patient undergoing treatment also serves as the cell donor, the transplanted cells (e.g., hematopoietic stem or progenitor cells) are less likely to undergo graft rejection. This stems from the fact that the infused cells are derived from the patient and express the same HLA class I and class II antigens as expressed by the patient. Alternatively, the patient and the donor may be distinct. In some embodiments, the patient and the donor are related, and may, for example, be HLA-matched. As described herein, HLA-matched donor-recipient pairs have a decreased risk of graft rejection, as endogenous T cells and NK cells within the transplant recipient are less likely to recognize the incoming hematopoietic stem or progenitor cell graft as foreign, and are thus less likely to mount an immune response against the transplant. Exemplary HLA-matched donor-recipient pairs are donors and recipients that are genetically related, such as familial donor-recipient pairs (e.g., sibling donor-recipient pairs). In some embodiments, the patient and the donor are HLA-mismatched, which occurs when at least one HLA antigen, in particular with respect to HLA-A, HLA-B and HLA-DR, is mismatched between the donor and recipient. To reduce the likelihood of graft rejection, for example, one haplotype may be matched between the donor and recipient, and the other may be mismatched.
Pharmaceutical Compositions and Dosing
[0338] In cases in which a patient is administered a population of cells that together express one or more therapeutic proteins of the disclosure, the number of cells administered may depend, for example, on the expression level of the desired protein(s), the patient, pharmaceutical formulation methods, administration methods (e.g., administration time and administration route), the patients age, body weight, sex, severity of the disease being treated, and whether or not the patient has been treated with agents to ablate endogenous pluripotent cells (e.g., endogenous CD34+ cells, hematopoietic stem or progenitor cells, or microglia, among others). The number of cells administered may be, for example, from 1×10.sup.6 cells/kg to 1×10.sup.12 cells/kg, or more (e.g., 1×10.sup.7 cells/kg, 1×10.sup.8 cells/kg, 1×10.sup.9 cells/kg, 1×10.sup.10 cells/kg, 1×10.sup.11 cells/kg, 1×10.sup.12 cells/kg, or more). Cells may be administered in an undifferentiated state, or after partial or complete differentiation into microglia. The number of pluripotent cells may be administered in any suitable dosage form.
[0339] Cells may be admixed with one or more pharmaceutically acceptable carriers, diluents, and/or excipients. Exemplary carriers, diluents, and excipients that may be used in conjunction with the compositions and methods of the disclosure are described, e.g., in Remington: The Science and Practice of Pharmacy (2012, 22nd ed.) and in The United States Pharmacopeia: The National Formulary (2015, USP 38 NF 33).
EXAMPLES
[0340] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure.
Example 1. Generation of a Pluripotent Stem Cell Expressing Functional NOD2 for the Treatment of Crohn's Disease
[0341] An exemplary method for making pluripotent cells (e.g., embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), or CD34+ cells) that express functional NOD2 is by way of transduction. Retroviral vectors (e.g., a lentiviral vector, alpharetroviral vector, or gammaretroviral vector) containing, e.g., a suitable promoter, such as a promoter described herein, and a polynucleotide encoding functional NOD2 can be engineered using vector production techniques described herein or known in the art. After the retroviral vector is engineered, the retrovirus can be used to transduce pluripotent cells (e.g., ESCs, iPSCs, or CD34+ cells) to generate a population of pluripotent cells that express functional NOD2.
[0342] Additional exemplary methods for making pluripotent cells that express functional NOD2 are transfection techniques. Using molecular biology procedures described herein and known in the art, plasmid DNA containing a promoter and a polynucleotide encoding functional NOD2 can be produced. For example, a nucleic acid encoding functional NOD2 may be amplified from a human cell line using PCR-based techniques known in the art, or a nucleic acid encoding functional NOD2 may be synthesized, for example, using solid-phase polynucleotide synthesis procedures. The nucleic acid and promoter can then be ligated into a plasmid of interest, for example, using suitable restriction endonuclease-mediated cleavage and ligation protocols. After the plasmid DNA is engineered, the plasmid can be used to transfect the pluripotent cells (e.g., ESCs, iPSCs, or CD34+ cells) using, for example, electroporation or another transfection technique described herein to generate a population of pluripotent cells that express the encoded protein(s).
Example 2. Functional NOD2 is an Important Contributor to the Proinflammatory Cytokine Response, and NOD2 Restoration Rescues the Ability of NOD2-Impaired Cells—Such as Those Observed in Crohn's Disease—to Mount a Cytokine Response
Introduction
[0343] This example describes the results of a series of experiments that were conducted with the aim of assessing the effects of NOD2 activation in various cell lines, including model systems of Crohn's disease in which functional NOD2 is depleted. As is described herein, Crohn's disease is a debilitating disorder characterized by the inability of endogenous cells to release inflammatory cytokines, particularly in response to muramyl dipeptide (MDP). As the results of these experiments demonstrate, functional NOD2 delivery to a system—such as by way of cell-based gene therapy, viral transduction, or a gene editing approach that inserts a functional NOD2 gene at a desired genetic locus—has the beneficial effect of restoring the ability of cells to mount an inflammatory immune response. In light of these results, NOD2 gene delivery represents a robust approach for the treatment of Crohn's disease.
Results
[0344] NOD2 Signaling in Healthy Cells Stimulates a Proinflammatory Cytokine Response
[0345] First, a set of experiments was conducted in order to demonstrate the ability of NOD2 activation to stimulate an inflammatory immune response in healthy human monocytes.
[0346] In addition, NOD2 activation was found to be capable of generating a robust inflammatory cytokine response even in the absence of priming. Peripheral blood CD14+ monocytes isolated from healthy human donors were treated with MDP to induce NOD2 signaling. NOD2-dependent cytokine production was then assayed in cell supernatants after 18-24 hours by flow based cytometric bead array analysis. As
[0347] A robust proinflammatory response to NOD2 activation was also observed in wild-type murine tissue-isolated and bone marrow-derived monocytes (
[0348] Depletion of Functional NOD2 Impairs the Proinflammatory Cytokine Response
[0349] Having established that NOD2 activation engenders an inflammatory cytokine response, another set of experiments was then conducted to assess the effects of NOD2 disruption on inflammatory cytokine release. As
[0350] To demonstrate the effects of NOD2 disruption in CD34+ hematopoietic stem cells (HSCs), peripheral blood-derived CD34+ cells isolated from healthy human donors were subject to targeted disruption of NOD2 by CRISPR-Cas9 gene editing (RNP+guideRNA nucleofection). Gene edited cells, NOD2 KO cells, and MOCK edited cells (receiving RNP only) were then cultured for 14 days in the presence of cytokines to promote differentiation towards monocyte/macrophage lineage committed cells. Cell cultures were then stimulated with MDP (0-100 μg/mL) for 18-24 hours and cell supernatants were assayed for IL-8 cytokine release by ELISA. As
[0351] The effects of NOD2 disruption in murine cells was also investigated. As
[0352] Restoration of NOD2 Rescues the Ability of Cells to Release Proinflammatory Cytokines
[0353] Having demonstrated that functional NOD2 is an important contributor to proinflammatory cytokine release, a series of experiments was then conducted to evaluate the ability of functional NOD2 delivery to restore the proinflammatory cytokine response in NOD2 deficient cells.
[0354] Not only was lentiviral transduction capable of restoring NOD2 expression, but this had the effect of improving inflammatory cytokine release. As
[0355]
[0356]
[0357] Significantly, the beneficial effects of functional NOD2 restoration are also observed in CD34+ HSCs. CD34+ cells isolated from mobilized peripheral blood of healthy human volunteers were transduced with lentiviral vectors (moi 10 & 50) generated to transfer WT or codon optimized NOD2 under the control of a myeloid lineage-specific promoter (CD11b promoter) or a constitutive promoter (EFS promoter) (
[0358] In addition to lentiviral delivery of NOD2, another approach for restoring NOD2 in a NOD2-deficient cell is by way of CRISPR-mediated gene editing, which can have the effect of inserting a functional NOD2 transgene at a desired genetic locus.
[0359] By substituting a functional NOD2 transgene for the GFP reporter used in
Conclusion
[0360] As the results of these experiments demonstrate, NOD2 is an important contributor to the ability of hematopoietic cells to mount a proinflammatory cytokine response, as the expression of NOD2 augments the release of proinflammatory cytokines and the disruption of NOD2 impairs the release of proinflammatory cytokines. Crohn's disease is associated with reduced NOD2 activity that, in turn, hinders the ability of cells to release proinflammatory cytokines. Importantly, the foregoing experiments demonstrate that the delivery of functional NOD2—whether by way of viral transduction, cell-based gene therapy, or a gene editing approach that inserts a functional NOD2 transgene at a desired genetic locus—rescues the ability of cells to mount a proinflammatory cytokine response.
Example 3. Administration of a Therapeutic Composition to a Patient Suffering from Crohn's Disease
[0361] According to the methods disclosed herein, a patient, such as a human patient, can be treated so as to reduce or alleviate symptoms of Crohn's disease and/or so as to target an underlying biochemical etiology of the disease. To this end, the patient may be administered, for example, a population of pluripotent cells, (e.g., ESCs, iPSCs, CD34+ cells) expressing functional NOD2. The population of pluripotent cells may be administered to the patient, for example, systemically (e.g., intravenously). The cells may be administered in a therapeutically effective amount, such as from 1×10.sup.6 cells/kg to 1×10.sup.12 cells/kg or more (e.g., 1×10.sup.7 cells/kg, 1×10.sup.8 cells/kg, 1×10.sup.9 cells/kg, 1×10.sup.10 cells/kg, 1×10.sup.11 cells/kg, 1×10.sup.12 cells/kg, or more).
[0362] Before the population of cells is administered to the patient, one or more agents may be administered to the patient to ablate the patient's endogenous hematopoietic cell population, for example, by administration of a conditioning agent described herein.
[0363] The success of the treatment may be monitored by way of various clinical indicators. Effective treatment of Crohn's disease using a composition of the disclosure may manifest, for example, as (i) sustained disease remission, such as sustained disease remission for at least one year; (ii) an observation that the patient no longer requires treatment with immunosuppressive agents, biologic agents, and/or corticosteroids; and/or (iii) an observation that the patient does not exhibit evidence of erosive disease in the gastrointestinal tract, as assessed by endoscopy and/or radiology.
OTHER EMBODIMENTS
[0364] Various modifications and variations of the described disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled in the art are intended to be within the scope of the disclosure.
[0365] Other embodiments are in the claims.