HOXA11as long non-coding RNA in inflammatory bowel disease

Abstract

Provided herein are gene replacement approaches to restore HOXA11AS in the colon for patients with IBD, e.g., UC. Further, since HOXA11os levels inversely correlate with disease severity this lncRNA can also be used as a sensitive colon specific disease relevant biomarker.

Description

[0001] H0XA1 IAS long non-coding RNA in Inflammatory Bowel Disease

[0002] CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 528.833, filed on July 25, 2023, and 63 / 532,249, filed on August 11, 2023. The entire contents of the foregoing are hereby incorporated by reference herein.

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing that has been submitted electronically as an XML file named “07917-0448W01.xml” The XML file, created on July 24, 2024, is 36,864 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0006] TECHNICAL FIELD

[0007] Provided herein are gene replacement approaches to restore HOXA11AS in the colon for patients with inflammatory bowel disease (IBD), e.g., ulcerative colitis (UC). Further, since HOXA1 los levels inversely correlate with disease severity methods are provided for using this IncRNA as a sensitive colon specific disease relevant biomarker.

[0008] BACKGROUND

[0009] Ulcerative colitis (UC) is a chronic inflammatory disease of the colon tissue. UC is a multifactorial disease with underlying causes attributed to dysregulated immune responses, defects in epithelial barrier integrity as well as genetic and environmental factors. To date the majority of UC related studies have focused on delineating the functional roles proteins play in the development of the disease. Although significant progress has been made in understanding the disease etiology, current UC treatments only alleviate symptoms and for many individuals fail to maintain long term remission of the disease. SUMMARY

[0010] In this study, we have identified a previously unannotated disease relevant transcript of a IncRNA in the HOX locus, termed H0XA11 os. H0XA11 os is uniquely expressed in the distal colon in both mice and humans where its expression inversely correlates with inflammation and disease seventy. Moreover, mice lacking HOXAllos are highly susceptible to colitis. HOXAllos is localized to the mitochondria under basal conditions where it interacts with complex I of the Electron Transfer Chain (ETC) that feeds into mitochondrial oxidative phosphorylation (OXPHOS). Mitochondria in HOXAllos deficient colonic myeloid cells are dysfunctional characterized by reduced levels of NAD and elevated mitochondrial reactive oxygen species (mtROS) which leads to the development of spontaneous intestinal inflammation. These studies identify a previously unannotated nuclear- encoded IncRNA that localizes to the mitochondria and regulates metabolic activity to maintain intestinal homeostasis.

[0011] Provided herein are methods for diagnosing inflammatory bowel disease (IBD) in a subject. The methods comprise providing a sample from the subject; detecting a level of a H0XA11 AS in the sample; and comparing the level of the H0XA11 AS in the sample to a reference level; wherein presence of a level of H0XA11AS below the reference level indicates that the subject has IBD.

[0012] Also provided herein are methods comprising: providing a sample from the subject; and detecting a level of aHOXAl IAS in the sample; and optionally comparing the level of the H0XA11 AS in the sample to a reference level.

[0013] Further provided herein are methods of treating IBD in a subject. The methods comprise providing a sample from the subject; detecting a level of aHOXAl IAS in the sample; comparing the level of the H0XA11 AS in the sample to a reference level; wherein presence of a level of H0XA1 IAS below the reference level indicates that the subject has IBD; and administering a treatment for IBD to the subject who has a level of H0XA11AS below the reference level.

[0014] In some embodiments, the sample comprises nucleic acids or cells from the intestine, e.g., distal colon, of the subject. In some embodiments, the sample comprises an intestinal biopsy or cytology brushing. In some embodiments, the HOXA1 IAS is at least 80%, 85%, 90%, 95%, or 99% identical to a HOXA11 AS-1 or HOXA11 AS-2 sequence shown in Table 2, e.g., SEQ ID NO: 5 or 6.

[0015] In some embodiments, detecting a level ofHOXAHAS comprises using RT- PCR.

[0016] Additionally, provided herein are methods for treating IBD in a subject. The methods comprise administering to the subject a H0XA11 AS nucleic acid to cells in the intestine of the subject, e.g., to myeloid cells in the intestine of the subject. Also provided are HOXA1 IAS nucleic acids as described herein for use in treating IBD in a subject. In some embodiments, the IBD is ulcerative colitis (UC).

[0017] In some embodiments, the HOXA1 IAS is at least 80%, 85%, 90%, 95%, or 99% identical to a HOXA11 AS-1 or HOXA11 AS-2 sequence shown in Table 2, e.g., SEQ ID NO: 5 or 6.

[0018] In some embodiments, the HOXA1 IAS nucleic acid is naked RNA or a viral vector comprising a sequence encoding HOXA1 IAS.

[0019] Additionally, provided herein are isolated nucleic acids encoding HOXA11AS, optionally wherein the HOXA1 IAS is at least 80%, 85%, 90%, 95%, or 99% identical to a HOXA11 AS-1 or HOXA11 AS-2 sequence shown in Table 2, e.g., SEQ ID NO: 5 or 6.

[0020] In some embodiments, the isolated nucleic acid is linked to or comprised within a nanoparticle, e.g., a lipid nanoparticle, optionally wherein the isolated nucleic acid is a HOXAHAS RNA.

[0021] Also provided herein are expression vectors comprising a sequence encoding HOXA11 AS operably linked to a promoter for expression of the HOXA11 AS, optionally wherein the HOXA11AS is at least 80%, 85%, 90%, 95%, or 99% identical to a HOXA11 AS-1 or HOXA11 AS-2 sequence shown in Table 2, e.g., SEQ ID NO:5 or 6. In some embodiments, the expression vector is a viral vector, optionally an AAV, adenovirus, or lentivirus.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0023] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0024] DESCRIPTION OF DRAWINGS

[0025] FIGs. 1A-H. HOXAllos / AS are highly abundant in the healthy colon and are downregulated in intestinal inflammation. Volcano plots representing differentially expressed IncRNAs in (A) resident macrophages and (B) infiltrated monocytes isolated from healthy and DSS treated mice. LncRNAs with p. value > 0.05 are presented as grey dots (Data are represented as the mean of n=2-5 from 3 independent experiments).24(C) Combined HOXAllos reads from Oxford Nanopore sequencing (top), reference genome Gencode vM26 annotation (middle) and HOXAllos transcripts (bottom) in the colon (n=2). (D) RT-qPCR analysis of murine colonic HOXAllos transcripts in the indicated mice. Gene expression levels were normalized to TBP (***P<0.001, Data represent the mean +SEM of n=6-10). (E) HOXA11AS TPM expression levels from aligned RNA sequencing26, 27, n=62- 228. HOXAllos / AS is predominantly expressed in the distal colon. (F) RT-qPCR analysis of HOXAllos mRNA in the indicated organs ofWT mice (n=4). (G) RT- qPCR analysis of HOXAllos mRNA in the indicated organs ofWT mice (n=4). (H) Representative images of single molecule RNAscope single-plex assay detecting endogenous HOXAllos in colon biopsies ofWT mice.

[0026] FIGs. 2A-H. HOXAllos is localized to the mitochondria. RT-qPCR analysis of HOXAllos, GAPDH and 7SK expression levels in the cytoplasm and nucleus of (A) LP cells and (B) lECs isolated from the distal colon ofWT mice presented as a % of total RNA (n=2). (C) Graphical summary of HOXA11 os-binding proteins identified by ChIRP followed by mass spectrometry. (D) RT-qPCR analysis of HOXAllos, TBP and COXII expression levels in the cytoplasm and purified mitochondria. (Data are represented as the mean of n=3 from 2 independent experiments). Representative image of single molecule RNAscope multi-plex assay detecting endogenous (E) HOXAllos and mitochondria and (F) RNA polymerase II and Peptidylprolyl Isomerase B in colon biopsies of WT mice. (G) Percentage of HOXAllos positive mitochondria in the distal colon of WT mice. (***P<0.001, Data represent the mean ±SEM of n=8-10 from 2-3 independent experiments). Arrows represent colocalization sites. (H) Representative images of single molecule RNAscope multi -plex assay detecting endogenous HOXAllos and mitochondria in the distal colon of HOXAllos-KO mice. Scale bar=10um.

[0027] FIGs. 3A-Q. HOXAllos-KO mice developed mild signs of spontaneous intestinal inflammation and are highly susceptible to colitis. Oxygen consumption rate of colonic cells isolated from HOXAllos-KO and WT littermate control mice at 4-months of age. (**P<0.01, Data represent the mean +SEM of n=2 from 5 independent experiments). RT-qPCR analysis of (B) IL-6, (C) IL-10 and (D) RANTES expression levels in colons isolated from the indicated mice at 4-month of age. (**P<0.01, ***P<0.001, Data represent the mean +SEM of n=7-8). (E) Schematic illustration of acute model of DSS-inded colitis. (F,J) Colon length, (G,K) percentage weight change and (H,L) stool appearance of HOXAllos-KO and WT littermate control mice treated with DSS for 7 days. (***P<0.001, Data represent the mean +SEM of n=2-8 from 6 independent experiments). (I,M) Pathological score of H&E-stained colon sections from HOXAllos-KO and WT littermate control mice treated with DSS for 7 days. (***P<0.001, Data represent the mean ±SEM of n=2-4 from 3 independent experiments). (N) Representative H&E-stained colon sections of the indicated mice. RT-qPCR analysis of (O) IL-6, (P) IL-10 and (Q) CXCL-10 expression levels in colons isolated from the indicated mice treated with DSS for 7 days. (*P<0.05, **P<0.01, Data represent the mean ±SEM of n=5-6 from 2 independent experiments).

[0028] FIGs. 4A-I. HOXAllos deficiency in the hematopoietic compartment promoted colitis. (A) Schematic illustration of bone marrow chimera experiments of the indicated irradiated (900R) mice reconstituted with 107bone marrow cells from donor mice, graphical summary of (B) weight loss, (C) colon length and (D) stool appearance of the indicated mice 4 days post DSS treatment. (*P<0.05,***P<0.001. Data represent the mean ±SEM of n=3-5 from 2 independent experiments). (F) Representative H&E-stained colon sections of the indicated mice. RT-qPCR analysis of (G) IL-6, (H) IL-10 and (I) CXCL-1 expression levels in colons isolated from the indicated mice 4 days post DSS treatment. (*P<0.05,** P<0.01. Data represent the mean +SEM of n=3-5 from 2 independent experiments).

[0029] FIGs. 5A-O. HOXAllos deficient immune cells displayed impaired complex I activity and dysfunctional mitochondria. (A) NAD+ / NADH ratio, (B) ATP nM concentration and (C) Mitochondrial ROS concentrations in CD45+LP immune cells isolated from the indicated mice at 4.5-months of age. (**P<0.01,***P<0.001, Data represent the mean +SEM of n=3 from 2 independent experiments). (D) RT-qPCR analysis of mitochondrial DNA as mDNA / nDNA ratio of colonic LP CD45+immune cells isolated from HOXAllos-KO and WT littermate control mice (*P<0.05,***P<0.001, Date represents the mean +SEM of n=7).

[0030] HOXAllos interacts with the complex I subunit, NDUFV1. (E) RT-qPCR analysis of HOXAllos and TBP expression levels in colonic HOXAllos-KO and WT LP cells of the indicated pulldowns. (***P<0.001, Data represent the mean +SEM of n=3 from 2 independent experiments). (F) Representative image of Western blotting of NDUFV1 and P-actin in colonic LP cells of HOXAllos-KO and WT littermate control mice with the indicated experimental groups. HOXAllos regulates complex I activity in LP myeloid cells. MitoTrackerGreen FMI analysis (G) and representative MFI images (H) of mitochondrial mass and MitoTrackerRed FMI analysis (I) and representative MFI images (J) of mitochondrial mass of colonic CD45+CDlb+myeloid cells isolated from HOXAllos-KO and WT littermate control mice with the indicated treatment. (**P<0.01, ***P<0.001, Date represents the mean +SEM of n=2-4 from 3 independent experiments). RT-qPCR analysis of (K) IL-6, (L) IL-1 p and (M) CXCL-10 expression levels in colonic myeloid cells isolated from the indicated mice (***P<0.001, Data represent the mean ±SEM of n=4). (N) Representative Western blotting image of mitochondrial markers in colonic LP CD45+cells isolated from HOXAllos-KO and WT littermate control mice. Equal loading was detected by measuring p-actin. n=5. (O) Representative images of colonic LP myeloid cells mitochondria obtained by transmission electron microscopy (TEM).

[0031] FIGs. 6A-I. Depletion of mtROS ameliorated colitis in HOXAllos-KO mice. (A) Schematic image illustration Rotenone experimental design. (B) Colon length, (C) percentage weight change, (D) stool appearance and (E) Pathological score of H&E-stained colon. (**P<0.01, ***P<0.001, Data represent the mean +SEM of n=3-6 from 2 independent experiments). (F) Representative H&E-stained colon sections of the indicated mice. RT-qPCR analysis of (G) IL-6, (H) IL- 1 p and (I) CXCL-10 expression levels in colons isolated from the indicated mice (**P<0.01, ***p<0.001, Data represent the mean +SEM of n=3-5 from 2 independent experiments).

[0032] FIGs. 7A-H. H0XA11AS is highly abundant in the healthy colon and is downregulated in ulcerative colitis. Related to Figure 1. (A) Combined

[0033] H0XA11 AS reads from Oxford Nanopore sequencing (top), reference. Gencode v39 annotation (middle) and H0XA11AS transcripts (bottom) in the colon. (B) RT-qPCR analysis of human colonic H0XA11AS transcripts in the indicated treatments. Gene expression levels were normalized to GAPDH (*P<0.05, Data represent the mean ±SEM of n=5-6). (C) Schematic illustration of colonic HOXAllos and H0XA11AS transcripts and the percentage sequence conservation calculated by PFAAT. See also Figure 12. (D) Representative image of Western blotting of Myc-tagged H0XA11 os- potential ORFs and positive control Myc-tagged Caspase-1 in HEK293T cells, n=2.

[0034] HOXAllos / AS is predominantly expressed in the distal colon. (E) H0XA11AS TPM expression level from aligned RNA sequencing79,80n=5-125. (F) RT-qPCR analysis of HOXAllos expression levels in CD45+ LP cells and CD45- lECs isolated from the distal colon. (n=8). (G) Representative image of single molecule RNAscope single-plex assay detecting endogenous HOXAllos (brown) in the indicated areas of colon biopsies of WT mice. (H) RT-qPCR analysis of HOXAllos expression levels in cells isolated from the distal colon of WT mice following the indicated treatments (Data are represented as mean ±SEM of n=2-4 pulled mice from 2 independent experiments).

[0035] FIGs. 8A-F. HOXAllos-KO mice exhibit comparable expression levels of the protein coding gene H0XA11 as WT mice. Related to Figure 1. (A) Schematic illustration of the CRISPR / Cas9 strategy to generate HOXAllos-KO mice (shown is SEQ ID NO: 12). (B) RT-qPCR analysis of HOXAllos transcript 1 and HOXAllos transcript 2 expression levels in the distal colon of the indicated mice. (***P<0.001, Data represent the mean ±SEM of n=4). (C) RTqPCR analysis of HOXAllos and H0XA11 protein expression levels in the distal colon of the indicated mice. (Data represent the mean ±SEM of n=6). (D) Immunoblot analysis of H0XA11 protein and b-actin in lysates generated from colon tissue from the indicated mice. (E) RT-qPCR analysis of HOXAllos and H0XA11 protein expression levels in the distal colon of the indicated mice treated with 2% DSS for 7 days. (Data represent the mean ±SEM of n=5). (F) Immunoblot analysis of H0XA11 protein and b-actin in lysates generated from colon tissue from the indicated mice treated with or without DSS.

[0036] FIGs. 9A-J. HOXAllos-KO mice demonstrated impaired recovery following DSS-induce colitis experiment. Related to Figure 3. (A) Representative images of immunohistochemistry staining of Ki 67 in distal colon biopsies collected from HOXAllos-KO and WT littermate control mice. (B) Quantification of Ki67 positive cells per field of view. (***P<0.001, Data represent the mean ±SEM of n=2- 17 from 2-3 independent experiments). Scale bar=100um. (C) Schematic image illustrating the recovery model of DSS-induced colitis. Graphical summary of (D) colon length, (E) stool appearance and (F) weight change and (G) pathological score of H&E-stained colon sections of HOXAllos-KO and WT littermate control mice 7 days post DSS treatment. (**P<0.05,***P<0.001, Data represent the mean ±SEM of n=2-8 from 3 independent experiments). (H) Representative H&E images of HOXAllos-KO and WT littermate control mice 7 days post DSS treatment. (I) pathological score of epithelial hyperplasia and pathological score of (J) epithelial erosion of of HOXAllos-KO and WT littermate control mice 7 days post DSS treatment. (*P<0.05,***P<0.001, Data represent the mean ±SEM of n=4-5 from 2 independent experiments).

[0037] FIGs. 10A-J S7. HOXAllos-KO mice are hyper susceptible to Salmonella Typhimuriuin-induced colitis. Related to Figure 3. (A) Colon length, (B) percentage weight change, (C) stool appearance and (D) Pathological score of H&E-stained colon sections from pretreated streptomycin HOXAllos-KO and WT littermate control mice infected with Salmonella Typhimurium. (*P<0.05, ***P<0.001, Data represent the mean ±SEM of n=5-7 from 3 independent experiments). (E) Representative H&E-stained colon sections of the indicated mice. RTqPCR analysis of (F) IL-6, (G) IL-1 b and (H) CXCL-10 expression levels in colons isolated from the indicated mice (*P<0.05, ***P<0.001, Data represent the mean ±SEM of n=5-7 from 3 independent experiments). HOXAllos function in the colon is independent on the microbiome. (I) RT-qPCR analysis of HOXAllos expression in the distal colon of WT mice treated for 4 weeks with a broad-spectrum antibiotic cocktail (ABX) or the indicated single antibiotic, ampicillin (Igr / L), neomycin (Igr / L), vancomycin (0.5gr / L) and metronidazole (Igr / L). (Data represent the mean ±SEM of n=5 from 2 independent experiments). (J) Principal coordinates analysis (PCoA) plot of whole genome shotgun sequencing (WGS) community profiles representing diversity based on genotype using the weighted UniFrac distance metnc, n=23-27.

[0038] FIGs. 11A-J. Rectal administration of complex I inhibitor to WT mice phenocopied colitis observed in HOXAllos-KO mice. Related to Figure 6. (A) Schematic image illustration Rotenone experimental design. (B) Colon length, (C) percentage weight change, (D) stool appearance and (E) pathological score of H&E- stained colon. (**P<0.01, ***P<0.001, Data represent the mean ±SEM of n=3-5 from 2 independent experiments). (F) Representative H&E-stained colon sections of the indicated mice. RT-qPCR analysis of (G) IL-6, (H) IL-lb and (I) CXCL-10 expression levels in colons isolated from the indicated mice (*P<0.05,**P<0.01, ***p<0.001, Data represent the mean ±SEM of n=3-5 from 2 independent experiments). Depletion of mtROS ameliorated colitis in HOXAllos-KO mice. (J) Representative H&E-stained colon sections of the indicated mice.

[0039] FIG. 12. Sequence conservation. Alignment of mouse and human sequences. Shown are SEQ ID NO: 1, nucleotides 1-1523 of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 19, and SEQ ID NO:20, in order of appearance.

[0040] DETAILED DESCRIPTION

[0041] New therapeutic targets are required to improve treatments and long-term outcomes for patients with Inflammatory Bowel Disease (IBD). While most studies have focused on delineating the functional roles of proteins in signaling pathways that initiate inflammation in the intestinal tract, a significant amount of genetic evidence suggests noncoding regions also plays important role in the regulation and progression of UC. Indeed, the majority of IBD-associated SNPs are located in noncoding genomic regions. LncRNAs account for the majority of RNA transcribed from noncoding genomic regions, accounting for a significant proportion of the human genome,1'3and have been implicated in the development of acute inflammation and chronic inflammatory diseases4 2To date over 16,000 IncRNAs have been annotated in the human genome13. Yet, the majority of these remain uncharacterized.

[0042] A small number of studies have revealed that IncRNA expression is associated with the pathogenesis of UC14, 15. LncRNAs have been implicated in UC through the regulation of miRNA expressionl6‘17or through modulation of intestinal epithelial barrier function18. Given that these studies have primarily relied on cell-based in vitro assays and gene expression profiling19'23, physiological functions of IncRNAs in the pathogenesis of UC remain to be defined in detail, and thus their physiological role in UC is still unknown.

[0043] Described herein is a nucl ear-encoded IncRNA, H0XA1 los, that localizes to mitochondria to promote mitochondrial function and maintain intestinal homeostasis. HOXAllos is a tissue specific IncRNA that is highly conserved between mice and humans. Given the unique expression profile of H0XA1 los in the distal colon, we performed all of our studies on physiologically relevant colon biopsies collected from mouse and human patients. LncRNAs are highly tissue specific29, and their annotation is poor and incomplete. Accurate annotation of IncRNAs is fundamental to define their biology. To overcome this limitation, we performed long read RNA sequencing on tissue biopsies which enabled the precise identification of the exact transcripts of HOXAl lAos expressed in the colon. This approach identified a previously unreported highly abundant transcript of HOXAllos.

[0044] Characterization of IncRNA subcellular localization and the proteins they interact with are central to understanding their function. Following identification of the specific H0XA1 los transcripts expressed in the colon, we designed H0XA11 os- specific probes to define the subcellular localization of HOXAl los. To date, IncRNAs have been shown to localize to the nucleus or cytoplasm. While studies have begun to interrogate the localization of IncRNAs to specific organelles, only a small portion of organelle-associated IncRNAs have been functionally characterized30.

[0045] Remarkably, H0XA1 los was localized to mitochondria within cells of the distal colon and interacted with mitochondria residing proteins in shared metabolic pathways. To study the physiological function of HOXAl los in the colon, we generated H0XA1 los deficient mice by introducing a minimal polyadenylation signal (pAS) downstream of the H0XA1 los transcription start site. This approach efficiently terminated the transcription of H0XA1 los in the distal colon while leaving the genomic locus intact. Interestingly, aged H0XA1 los deficient mice demonstrated impaired mitochondrial OXPHOS in cells of the distal colon and impaired turnover of epithelial cells. HOXAllos deficient mice developed mild spontaneous intestinal inflammation and were hyper susceptible to experimental models of colitis. These data suggest that HOXAllos functions to restrict age associated susceptibility to intestinal inflammation by maintaining optimal functional mitochondria in cells of the distal colon.

[0046] Mitochondrial dysfunction has long been associated with intestinal inflammation. Indeed, mice carrying mitochondrial polymorphisms associated with higher OXPHOS activity and ATP production are protected from colitis33. Mitochondrial dysfunction has also been associated with human UC. A decrease in OXPHOS gene expression39'41> 36, deficiencies in electron transport chain activity and elevated mtROS have all been reported in patients with UC37'39. These data suggest that mitochondnal dysfunction is a key pathogenic feature of UC. mtROS plays an important role in host defense. Yet, under pathological conditions, high mtROS levels can lead to oxidative damage and inflammation60’61. Furthermore, excessive mtROS levels can triggers colitis, whereas mtROS inhibitors alleviate colitis in mice62.

[0047] HOXAllos was found at high levels in both the hematopoietic and non- hematopoietic cells of the distal colon and deficiency of H0XA1 los in both cellular compartments resulted in impaired complex I activity, characterized by decreased NAD production and elevated mtROS. Yet, bone marrow chimera experiments revealed that complex I deficiency in the hematopoietic compartment but not the non- hematopoietic compartment was the cause of colitis susceptibility. This suggest that immune cells in the colon may be more susceptible to dysfunctional complex I activity when compared to lECs due to slower cellular turnover or due to different metabolic requirements that are unique to myeloid cells. H0XA1 los-KO immune cells displayed dysregulation of the complex I subunit, NDUFV1 and a decrease in the protein levels of markers of mitochondrial mass and biogenesis. H0XA1 los-KO immune cells also displayed elevated levels of the mitophagy regulator PARKIN. Rotenone treated colonic cells from WT mice mirrored the effects of HOXAl los deficiency on these dynamic markers. Immune cells isolated from H0XA1 los-KO mice demonstrated comparable protein levels to cells isolated from H0XA1 los-KO mice treated with rotenone. These data strongly support our conclusions that HOXAllos exhibits its function via the regulation of complex I in colonic immune cells.

[0048] Through the use of bone marrow chimeras, we defined immune cells as key drivers of colitis susceptibility in H0XA1 los-KO mice. H0XA1 los-KO myeloid cells displayed decreased A'Pm and a disrupted mitochondrial ultrastructure. Comparable levels of ATV were observed in H0XA1 los-KO myeloid cells treated with or without rotenone, further supporting the role of complex I impairment as the key mediator of colitis in H0XA1 los deficient mice. Rotenone treatment of WT mice phenocopied the colitis phenotype observed in H0XA1 los-KO mice. Additionally, inhibition of mtROS ameliorated colitis in H0XA1 los-KO mice. Collectively these findings underscore the importance of H0XA1 los as a regulator of complex I activity in colonic myeloid cells. HOXAl los likely regulates complex 1 activity by regulating protein-protein interactions, enhancing substrate binding or through regulation of catalytic activity.

[0049] In summary, we have identified a previously unknown mechanism by which the IncRNA HOXAl los regulates cell metabolism to maintain intestinal homeostasis. The unique regulatory function of H0XA1 los in the colon emphasizes the importance of noncoding genomic regions in the regulation of UC. This study provides the first characterization of how a IncRNA regulates cell metabolism at a tissue level and how its dysregulation can impact chronic diseases, such as UC. HOXAllos is abundant in the healthy colon and significantly reduced in colitis.

[0050] Thus, provided herein are gene replacement approaches to restore H0XA11AS (the human homolog of H0XA1 los) in the colon for patients with IBD, e.g., with UC. Further, since HOXAl los levels inversely correlate with IBD / UC disease severity this IncRNA can also be used as a sensitive colon specific disease relevant biomarker.

[0051] Methods of Diagnosis

[0052] Included herein are methods for diagnosing an inflammatory bowel disease in a subject. The methods rely on detection of levels of H0XA11AS. The methods include obtaining a sample from a subject, and evaluating the presence and / or level of H0XA11AS in the sample, and comparing the presence and / or level with one or more references, e.g., a control reference that represents a normal level of H0XA11AS, e.g., a level in an unaffected subject, and / or a disease reference that represents a level of the proteins associated with IBD, e.g., a level in a subject having IBD, e.g., CD or UC.

[0053] As used herein the term “sample”, when referring to the material to be tested for the presence of a biological marker using the method of the invention, includes inter aha tissue (e.g., from a biopsy), intestinal brushings, and stool samples. Various methods are well known within the art for the identification and / or isolation and / or purification of a biological marker from a sample. An “isolated” or “punfied” biological marker is substantially free of cellular material or other contaminants from the cell or tissue source from which the biological marker is derived, i.e., partially or completely altered or removed from the natural state through human intervention. For example, nucleic acids contained in the sample can be isolated according to standard methods, for example using lytic enzymes, chemical solutions, or isolated by nucleic acid-binding resins following the manufacturer’s instructions.

[0054] The presence and / or level of a H0XA11 AS nucleic acid can be evaluated using methods known in the art, e.g., using polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), quantitative or semi-quantitative real-time RT-PCR, digital PCR i.e. BEAMing ((Beads, Emulsion, Amplification, Magnetics) Diehl (2006) Nat Methods 3:551-559) ; RNAse protection assay; Northern blot; various types of nucleic acid sequencing (Sanger, pyrosequencing, NextGeneration Sequencing); fluorescent in-situ hybridization (FISH); or gene array / chips) (Lehninger Biochemistry (Worth Publishers, Inc., current addition; Sambrook, et al, Molecular Cloning: A Laboratory Manual (3. Sup.rd Edition, 2001); Bernard (2002) Clin Chem 48(8): 1178-1185; Miranda (2010) Kidney International 78: 191-199; Bianchi (2011) EMBO Mol Med 3:495-503; Taylor (2013) Front. Genet. 4: 142; Yang (2014) PLOS One 9(1 l):el 10641); Nordstrom (2000) Biotechnol. Appl. Biochem. 31(2):107-l 12; Ahmadian (2000) Anal Biochem 280: 103-110. In some embodiments, high throughput methods, e.g., protein or gene chips as are known in the art (see, e.g., Ch. 12, Genomics, in Griffiths et al., Eds. Modem genetic Analysis, 1999, W. H. Freeman and Company; Ekins and Chu, Trends in Biotechnology, 1999, 17:217-218; MacBeath and Schreiber, Science 2000, 289(5485): 1760-1763; Simpson, Proteins and Proteomics: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 2002; Hardiman, Microarrays Methods and Applications: Nuts & Bolts, DNA Press, 2003), can be used to detect the presence and / or level of HOXA1 IAS. Measurement of the level of a biomarker can be direct or indirect. For example, the abundance levels of H0XA1 IAS can be directly quantitated.

[0055] RT-PCR can be used to determine levels of HOXA1 IAS. The first step in expression profiling by RT-PCR is the reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction (Ausubel et al (1997) Current Protocols of Molecular Biology, John Wiley and Sons). To minimize errors and the effects of sample-to-sample variation, RT-PCR is usually performed using an internal standard, which is expressed at constant level among tissues, and is unaffected by the experimental treatment. Housekeeping genes are most commonly used.

[0056] Gene arrays are prepared by selecting probes which comprise a polynucleotide sequence, and then immobilizing such probes to a solid support or surface. For example, the probes may comprise DNA sequences, RNA sequences, co-polymer sequences of DNA and RNA, DNA and / or RNA analogues, or combinations thereof. The probe sequences can be synthesized either enzymatically in vivo, enzymatically in vitro (e.g. by PCR), or non-enzymatically in vitro.

[0057] In some embodiments, the presence and / or level of H0XA1 IAS is comparable to the presence and / or level of the protein(s) in the disease reference, and the subject has one or more symptoms associated with IBD then the subject has IBD. In some embodiments, the subject has no overt signs or symptoms of IBD, but the presence and / or level of one or more of the proteins evaluated is comparable to the presence and / or level of the protein(s) in the disease reference, then the subject has an increased risk of developing IBD. In some embodiments, once it has been determined that a person has IBD, or has an increased risk of developing IBD, then a treatment, e.g., as known in the art or as described herein, can be administered.

[0058] Suitable reference values can be determined using methods known in the art, e.g., using standard clinical trial methodology and statistical analysis. The reference values can have any relevant form. In some cases, the reference comprises a predetermined value for a meaningful level of H0XA11AS, e.g., a control reference level that represents a normal level of H0XA11AS, e.g., a level in an unaffected subject or a subject who is not at risk of developing a disease described herein, and / or a disease reference that represents a level of the proteins associated with conditions associated with IBD, e.g., a level in a subject having IBD (e.g., CD or UC).

[0059] The predetermined level can be a single cut-off (threshold) value, such as a median or mean, or a level that defines the boundaries of an upper or lower quartile, tertile, or other segment of a clinical trial population that is determined to be statistically different from the other segments. It can be a range of cut-off (or threshold) values, such as a confidence interval. It can be established based upon comparative groups, such as where association with risk of developing disease or presence of disease in one defined group is a fold higher, or lower, (e.g., approximately 2-fold, 4-fold, 8-fold, 16-fold or more) than the risk or presence of disease in another defined group. It can be a range, for example, where a population of subjects (e.g., control subjects) is divided equally (or unequally) into groups, such as a low-risk group, a medium-risk group and a high-risk group, or into quartiles, the lowest quartile being subjects with the lowest risk and the highest quartile being subjects with the highest risk, or into n-quantiles (i.e., n regularly spaced intervals) the lowest of the n-quantiles being subjects with the lowest risk and the highest of the n- quantiles being subjects with the highest risk.

[0060] In some embodiments, the predetermined level is a level or occurrence in the same subject, e.g., at a different time point, e g., an earlier time point.

[0061] Subjects associated with predetermined values are typically referred to as reference subjects. For example, in some embodiments, a control reference subject does not have a disorder described herein (e.g., IBD).

[0062] A disease reference subject is one who has (or has an increased risk of developing) IBD. An increased risk is defined as a risk above the risk of subjects in the general population.

[0063] Thus, in some cases the level of H0XA11 AS in a subject being less than or equal to a reference level of H0XA1 IAS is indicative of a clinical status (e.g., indicative of a disorder as described herein, e.g., IBD. In other cases the level of H0XA11AS in a subject being greater than or equal to the reference level of H0XA11AS is indicative of the absence of disease or normal risk of the disease. In some embodiments, the amount by which the level in the subject is the less than the reference level is sufficient to distinguish a subject from a control subject, and optionally is a statistically significantly less than the level in a control subject. In cases where the level of H0XA1 IAS in a subject being equal to the reference level of H0XA11AS, the “being equal” refers to being approximately equal (e.g., not statistically different). In some embodiments, the level ofHOXAHAS is compared to a range of reference levels that represent varying severities of IBD, wherein lower levels of H0XA11AS correlate with more severe forms of IBD.

[0064] The predetermined value can depend upon the particular population of subjects (e.g., human subjects) selected. For example, an apparently healthy population will have a different ‘normal’ range of levels of H0XA1 IAS than will a population of subjects which have, are likely to have, or are at greater risk to have, a disorder described herein. Accordingly, the predetermined values selected may take into account the category (e.g., sex, age, health, risk, presence of other diseases) in which a subject (e.g., human subject) falls. Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art.

[0065] In characterizing likelihood, or risk, numerous predetermined values can be established.

[0066] Methods of Treatment

[0067] The methods described herein include methods for the treatment of disorders associated with inflammation in the colon. In some embodiments, the disorder is inflammatory bowel disease (IBD). IBD is a collective term that describes conditions associated with chronic or recurring immune response and inflammation of the gastrointestinal (GI) tract. The two most common inflammatory bowel diseases are ulcerative colitis (UC) and Crohn’s disease (CD). Both are marked symptoms of IBD, for example, severe diarrhea, abdominal pain, fatigue, and weight loss. IBD and other gastrointestinal inflammatory diseases can be debilitating and sometimes lead to lifethreatening complications.

[0068] As used in this context, to “treat” means to ameliorate at least one symptom of the disorder associated with inflammation in the colon. Often, these disorders result in diarrhea, abdominal pain, fatigue, and weight loss; thus, administration of a therapeutically effective amount of a treatment can result in one or more of a reduction in diarrhea, abdominal pain, fatigue, and weight loss and a return or approach to normal stools, cessation of pain, lessening of fatigue, and weight gain or stabilization.

[0069] In some embodiments, the methods include administering a therapeutically effective amount of a nucleic acid encoding H0XA11 AS as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment.

[0070] In some embodiments, the methods include administering a treatment for IBD as known in the art to a subject identified as being in need thereof using a method described herein. Such treatments can include administration of aminosalicylates (e.g., sulfasalazine (SASP) and other types of 5-aminosalicylic acid (5-ASA) drugs), corticosteroids (CSs, e.g., budesonide, beclomethasone dipropionate), immunomodulators (e.g., thiopurines (TPs), methotrexate (MTX), calcineunn inhibitors, and Janus Kinase (JAK) inhibitors), biologies (e.g., inhibitors of pro- inflammatory cytokines TNF-a (e.g., infliximab (IFX), adalimumab (ADA), golimumab) and IL- 12 / IL-23 (e.g., ustekinumab, mirikizumab, risankizumab, and integrin antagonists (e.g., vedolizumab, etrolizumab, carotegrast Methyl (AJM300), and PF-00547659), and oral small molecules (e.g., JAK inhibitors such as tofacitinib, filgotinib, upadacitinib, deucravacitinib; sphingosine- 1 -phosphate receptor modulators and agonists, e.g., ozanimod or etrasimod). In some embodiments, the treatment includes resection, e.g., laparoscopic resection; apheresis therapy (e.g., Adsorptive granulocyte / monocyte apheresis (GMA)); antibiotics, probiotics, prebiotics, postbiotics, synbiotics, and fecal microbiota transplantation (FMT); and stem cell therapy comprising administration of haematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), and intestinal stem cells (ISCs). See, e.g., Cai et al., Front Med (Lausanne). 2021; 8: 765474.

[0071] HOXA11AS Nucleic Acids

[0072] The HOXA11AS nucleic acids described herein, e.g., nucleic acids encoding H0XA11 AS, can be incorporated into a gene construct to be used as a part of a gene therapy protocol. The invention includes targeted expression vectors for in vivo transfection and expression of HOXA11AS in particular cell types, especially myeloid cells of the gut. Expression constructs of such components can be administered in any effective carrier, e.g., any formulation or composition capable of effectively delivering the component gene to cells in vivo. Approaches include insertion of the gene in viral vectors, including recombinant retroviruses, adenovirus, adeno- associated virus, lentivirus, and herpes simplex virus-1, or recombinant bacterial or eukaryotic plasmids. Viral vectors transfect cells directly; plasmid DNA can be delivered naked or with the help of, for example, cationic liposomes (lipofectamine) or derivatized (e.g., antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPCL precipitation carried out in vitro or in vivo.

[0073] A preferred approach for in vivo introduction of nucleic acid into a cell is by use of a viral vector containing nucleic acid, e.g., a cDNA. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells that have taken up viral vector nucleic acid.

[0074] Retrovirus vectors and adeno-associated vims vectors can be used as a recombinant gene delivery system for the transfer of exogenous genes in vivo particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. The development of specialized cell lines (termed “packaging cells”) which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are characterized for use in gene transfer for gene therapy purposes (for a review see Miller, Blood 76:271 (1990)). A replication defective retrovirus can be packaged into virions, which can be used to infect a target cell through the use of a helper virus by standard techniques.

[0075] Protocols for producing recombinant viruses and for infecting cells in vitro or in vivo with such viruses are known in the art; examples can be found in Ausubel, et al., eds., Gene Therapy Protocols Volume 1: Production and In Vivo Applications of Gene Transfer Vectors, Humana Press, (2008), pp. 1-32; Ghosh et al., Viral Vector Systems for Gene Therapy: A Comprehensive Literature Review of Progress and Biosafety Challenges, Applied Biosafety. Mar 2020. 7-18; and other laboratory manuals.

[0076] Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are known to those skilled in the art. Examples of suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include T'Crip. T'Cre. 2 and Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and / or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Feriy et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640- 7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89: 10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; U.S. Patent No. 4,868,116; U.S. Patent No. 4,980,286; PCT Application WO 89 / 07136; PCT Application WO 89 / 02468; PCT Application WO 89 / 05345; and PCT Application WO 92 / 07573).

[0077] Another viral gene delivery system useful in the present methods utilizes adenovirus-derived vectors. The genome of an adenovirus can be manipulated, such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See, for example, Berkner et al., BioTechniques 6:616 (1988); Rosenfeld et al., Science 252:431-434 (1991); and Rosenfeld et al., Cell 68: 143-155 (1992). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, or Ad7 etc.) are known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances, in that they are not capable of infecting nondividing cells and can be used to infect a wide variety of cell types, including epithelial cells (Rosenfeld et al., (1992) supra). Furthermore, the vims particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situ, where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al., supra; Haj-Ahmand and Graham, J. Virol. 57:267 (1986). Adenoviral delivery of nucleic acids to the intestine has been reported; see, e.g., Cheng et al. Hum Gene Ther. 1997;8:755-64; Foreman et al. Hum Gene Ther. 1998;9: 1313-21; Hogaboam et al., J Clin Invest. 1997 Dec 1; 100(11): 2766-76.

[0078] A viral vector system particularly useful for delivery of nucleic acids is the adeno-associated virus (AAV). Adeno-associated virus is a naturally occurring defective vims that requires another vims, such as an adenovims or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al., Curr. Topics in Micro and Immunol. 158:97-129 (1992)). AAV vectors efficiently transduce various cell types and can produce long-term expression of transgenes in vivo. Although AAV vector genomes can persist within cells as episomes, vector integration has been observed (see for example Deyle and Russell, Curr Opin Mol Ther. 2009 Aug; 11(4): 442-447; Asokan et al., Mol Ther. 2012 April; 20(4): 699-708; Flotte et al., Am. J. Respir. Cell. Mol. Biol. 7:349-356 (1992); Samulski et al., J. Virol. 63:3822-3828 (1989); and McLaughlin et al., J. Virol. 62: 1963-1973 (1989)). AAV vectors, such as AAV2, have been extensively used for gene augmentation or replacement and have shown therapeutic efficacy in a range of animal models as well as in the clinic; see, e.g., Mingozzi and High, Nature Reviews Genetics 12, 341-355 (2011); Deyle and Russell, Curr Opin Mol Ther. 2009 Aug; 11(4): 442-447; Asokan et al., Mol Ther. 2012 April; 20(4): 699-708. AAV vectors containing as little as 300 base pairs of AAV can be packaged and can produce recombinant protein expression.

[0079] Thus, in some embodiments, the HOXA1 IAS nucleic acid is present in a vector for gene therapy, such as an AAV vector. In some instances, the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAV11, and AAV12. The use of AAV vectors to deliver constructs for expression in the intestinal tract has been described; see, e.g., Shao et al., J Pediatr Gastroenterol Nutr 43: 168-179 (oral administration of AAV2); Polyak et al., Dig Dis Sci. 2008 May; 53(5): 1261-1270 (Gene delivery to intestinal epithelial cells in vitro and in vivo with recombinant AAV11, 2 and 5 / AAV pseudotypes 2 / 1, 2 / 2, and 2 / 5); During et al., Nature Medicine 4:1131-1135 (1998) (orally administered AAV for gene therapy of lactose intolerance); Buckinx and Timmermans, Histochemistry and Cell Biology' volume 146, pages709-720 (2016); and Polyak et al., Am J Physiol Gastrointest Liver Physiol. 2012 Feb l;302(3):G296- 308 (AAVrhlO / AAV pseudotypes 4, 7, 8, 9, and 10).

[0080] An AAV vector as described herein can be a pseudotyped vector. Pseudotyping provides a mechanism for modulating a vector’s target cell population. For instance, pseudotyped AAV vectors can be utilized in various methods described herein. Pseudotyped vectors are those that contain the genome of one vector, e.g., the genome of one AAV serotype, in the capsid of a second vector, e.g., a second AAV serotype. Methods of pseudotyping are well known in the art. For instance, a vector may be pseudotyped with envelope glycoproteins derived from Rhabdovirus vesicular stomatitis virus (VSV) serotypes (Indiana and Chandipura strains), rabies virus (e.g., various Evelyn-Rokitnicki-Abelseth ERA strains and challenge virus standard (CVS)), Lyssavirus Mokola virus, a rabies-related virus, vesicular stomatitis virus (VSV), Mokola virus (MV), lymphocytic choriomeningitis virus (LCMV), rabies virus glycoprotein (RV-G), glycoprotein B type (FuG-B), a variant of FuG-B (FuG- B2) or Moloney murine leukemia virus (MuLV). A virus may be pseudotyped for transduction of one or more neurons or groups of cells. In addition, the capsid can be altered to include one or more peptides that increase expression in the CNS, see, e.g., Yao et al., Nat Biomed Eng. 2022 Oct 10. doi: 10. 1038 / s41551-022-00938-7;

[0081] Chatterjee et al., Gene Ther. 2022 Jun;29(6): 390-397; Meng et al., Mol Ther Methods Clin Dev. 2021 Feb 27;21:28-41; Zhang et al., Biomaterials. 2022 Feb;281: 121340; Gray, Cell Gene Ther. Insights 5, 1361-1368 (2019); Nonnenmacher et al., Mol.

[0082] Ther. Methods Clin. Dev. 20, 366-378 (2021).

[0083] Without limitation, illustrative examples of pseudotyped AAV vectors include recombinant AAV2 / 1, AAV2 / 2, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV9, AAVrhlO, AAV 11, and AAV12 serotype vectors. It is known in the art that such vectors may be engineered to include a transgene encoding a human protein or other protein. In particular instances, the present disclosures can include a pseudotyped AAV9 or AAVrhlO viral vector including a nucleic acid as disclosed herein. See Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003.

[0084] In some instances, a particular AAV serotype vector may be selected based upon the intended use, e.g., based upon the intended route of administration.

[0085] AAV vectors preferably include inverted terminal repeats (ITRs); promoters, enhancers (e.g., CMV enhancer), other cis-regulatory elements, and / or capsid serotype variants that control and drive expression of HOXA11 AS. With regard to promoters, vectors can include promoters that drive expression in many cell ty pes (e.g., human P-actin, human elongation factor-la, chicken -actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV), simian virus 40 (SC40), herpes simplex virus thymidine kinase (HSVTK), PGK, CAG, sCAG, or CASI promoters) or specifically in myeloid cells, e.g., mfap4 promoter, CSF1R macrophage promoter CD68 promoter, or a synthetic promoter derived from elements of transcription factor genes (Levin et al., Gene Therapy 19: 1041-1047 (2012)); or in epithelial cells of the intestinal tract, e.g., T3(b) promoter (Aihara et al., FEBS Lett. 1999 Dec 10;463(l -2): 185-8); human intestinal fatty acid binding protein promoter (HIFABP), rat intestinal fatty acid binding protein promoter (RIFABP), human mucin-2 promoter (HMUC2), human lysozyme promoter (HLY), human sucrase- isomaltase enhancer (HSI); and rat intestinal trefoil factor (RITF)(Ya-Feng et al., J Anim Sci Biotechnol. 2012; 3(1): 32). Other cis-regulatory elements can include posttranscriptional regulatory elements; 2A enhancers; polyadenylation sequences; and / or an intron. Posttranscriptional regulatory elements can include HBV Posttranscriptional Regulatory Element (HPRE), woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or variants thereof (e.g., WPRE2, WPRE3 see, e.g., Kalev-Zylinska ML, During MJJ Neurosci. 2007 Sep 26; 27(39): 10456-67; Zanta-Boussif et al., Gene Therapy (16): 605-619 (2009); Choi et al., Mol Brain. 7:17 (2014); US6136597).

[0086] Various methods for application of AAV vector constructs in gene therapy are known in the art, including methods of modification, purification, and preparation for administration to human subjects (see, e.g., Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003). In addition, AAV based gene therapy targeted to cells of the CNS has been described (see, e.g., U.S. patents 6,180,613 and 6,503,888). High titer AAV preparations can be produced using techniques known in the art, e.g., as described in U.S. Pat. No. 5,658,776

[0087] A vector construct refers to a polynucleotide molecule including all or a portion of a viral genome and a transgene. In some instances, gene transfer can be mediated by a DNA viral vector, such as an adenovirus (Ad) or adeno-associated vims (AAV). Other vectors useful in methods of gene therapy are known in the art. For example, a construct as disclosed herein can include an alphavirus, herpesvirus, retrovirus, lentivirus, or vaccinia vims.

[0088] Also provided herein are methods of making the AAV, as well as host cells comprising the AAV. Methods for obtaining recombinant AAVs having a desired capsid protein are well known in the art. (See, e.g., US 2003 / 0138772 and WO 2019 / 200286, the contents of which are incorporated herein by reference in their entirety). Typically the methods involve culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; a recombinant AAV vector composed of AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the recombinant AAV vector into the AAV capsid proteins.

[0089] In addition to viral transfer methods, such as those illustrated above, non-viral methods can also be employed to cause expression of a HOXAHAS nucleic acid in the tissue of a subject. Typically non-viral methods of gene transfer rely on the normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In some embodiments, non-viral gene delivery systems can rely on endocytic pathways for the uptake of the subject gene by the targeted cell. Exemplary gene delivery systems of this type include liposomal derived systems, poly -lysine conjugates, and artificial viral envelopes. Other embodiments include plasmid injection systems such as are described in Meuli et al., J. Invest. Dermatol. 116(1): 131-135 (2001); Cohen et al., Gene Then 7(22): 1896-905 (2000); or Tam et al., Gene Then 7(21): 1867-74 (2000).

[0090] For example, nucleic acids comprising DNA or mRNA encoding HOXA11AS, or HOXA1 IAS RNA, can be delivered to cells directly, e.g., encapsulated in a delivery vehicle, e.g., lipids and lipid nanoparticles (LNP); polymers and polymer-based nanoparticles (see Paunovska et al., Nature Reviews Genetics volume 23, pages 265-280 (2022)). Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present disclosure into suitable host cells. In particular, the AAV or HOXA1 IAS nucleic acids can be formulated for delivery encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle (e.g., conjugated to a nanoparticle) or the like. See, e.g., Tanaka et al., Heliyon. 2018 Dec; 4(12): e00959.

[0091] In some embodiments, a HOXA1 IAS nucleic acid is entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins), which can be tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al., No Shinkei Geka 20:547-551 (1992); PCT publication W091 / 06309; Japanese patent application 1047381; and European patent publication EP-A-43075).

[0092] In clinical settings, the HOXA11AS gene delivery systems can be introduced into a subject by any of a number of methods, each of which is familiar in the art. For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g., by intravenous injection, oral administration, or and specific transduction of the protein in the target cells will occur predominantly from specificity of transfection, provided by the gene delivery vehicle, cell-type or tissuetype expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof. In other embodiments, initial delivery of the recombinant gene is more limited, with introduction into the subject being quite localized. For example, the gene delivery vehicle can be introduced orally, luminally, by catheter (see, e.g., Prieto et al., Gut. 2003 May; 52(Suppl 2): ii49— ii54).

[0093] In some embodiments, the pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent or can comprise a slow release matrix in which the gene delivery vehicle is embedded. Alternatively, where the complete gene delivery system can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can comprise one or more cells, which produce the gene delivery' system.

[0094] Also provided herein are compositions and formulations comprising the HOXA11AS nucleic acids and / or HOXA 11 AS encoding vectors, e.g., in a sterile carrier. Formulation of pharmaceutically-acceptable excipients and carrier solutions is w ell-known to those of skill in the art. As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.

[0095] Exemplary sequences

[0096] In some embodiments, the sequence of a H0XA1 IAS nucleic acid used in a composition or method described herein is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to a sequence set forth herein, e.g., a sequence shown in Table 2, e.g., SEQ ID NO:5 or 6. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non- homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identify between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0097] The comparison of sequences and determination of percent identify between two sequences can be accomplished using a mathematical algorithm. For example, the percent identify between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available on the world wide web at gcg.com), using the default parameters, e.g., a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0098] EXAMPLES

[0099] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0100] Methods and Materials

[0101] The following materials and methods were used in the Examples set forth below.

[0102] EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

[0103] Animal studies. All animal experiments were approved by the Institutional Animal Care Use Committees at the UMass Chan Medical School. Animal were kept in specific pathogen free (SPF) environment. HOXA1 los mice were generated on the C57BL / 6 background by the insertion of synthetic poly adenylated, poly(A), site33to the first exon using CRISPR-Cas9 technology (Extended Data Fig 3A). The design and synthesis of gRNAs were kindly provided by Prof. Jorge Henao-Mejia from the University of Pennsylvania. Genotyping of H0XA1 los-KO mice was performed in Transnetyx using TaqMan™ qPCR assay.

[0104] For the identification of H0XA1 los-KO in mice, the following primers were used.

[0105] H0XA1 los-KO Forward: GCTCAGCTACATGGTCCTGGTC (SEQ ID NO:13)

[0106] H0XA1 los-KO Reverse: TCCGGGACCTGAAGCTATACA (SEQ ID NO: 14)

[0107] H0XA1 los-KO Reporter: CATTAGATCTGTGTGTTGGTT (SEQ ID NO:15)

[0108] For the identification of H0XA1 los-WT in mice, the following primers were used.

[0109] H0XA1 los-WT Forward GCAGCTCTCCTTTGCTCAGCTACA (SEQ ID NO: 16)

[0110] H0XA1 los-WT Reverse CTCTCTTGCCGCCCTCA (SEQ ID NO: 17)

[0111] H0XA1 los-WT Reporter CACTCGTGGACCAGG (SEQ ID NO: 18)

[0112] Mice were generated by crossing heterozygous male mice with heterozygous littermate control female mice. Co-housed WT littermate control mice were used throughout the study. No gender bias was observed for intestinal inflammation and both male and female mice were used. Group sizes are specified in the figure legends. Animals were used solely for experiments described in this study and were not subjected to any treatment unless mentioned otherwise. Mice were co-housed with no more than 5 mice per cage.

[0113] Human subjects. Intestine biopsies collected from de-identified patients with ulcerative colitis and healthy individuals were used in this study. Biopsies samples were collected and stored in -80°C until used.

[0114] DSS-induced acute colitis. For acute model of colitis, H0XA1 los-KO and WT littermate control mice were co-house and treated with 2% DSS in the drinking water for 7 days. For recovery model of colitis, H0XA1 los-KO and WT littermate control mice were treated with 1.5% DSS in the drinking water for 5 days followed by regular water for 7 days.

[0115] Salmonella-induced colitis. H0XA1 los-KO and WT littermate control mice were pretreated with 20mg streptomycin for 1 day following by administration of S. typhimurium (SL1344) by gavage (1.8*10A8 CFU). Colon samples were extracted 4 days post infection.

[0116] MitoQ in vivo model. HOXAllos-KO and WT litermate controls were co-house and treated with 2% DSS in the drinking water for 7 days. An amount of 5mg / kg MitoQ was administrated by intraperitoneal every other day during the duration of DSS treatment.

[0117] Rotenone in vivo model. H0XA1 los-KO and WT littermate control were co-house and treated with 2% DSS in the drinking water for 7 days. An amount of Img / kg of rotenone was rectally every other day during the duration of DSS treatment.

[0118] Immunobloting. For immunoblot analysis cells were lysed in NP-40 lysis buffer (50mM Tris-HCl, pH 7.4, containing 150mM NaCl, 0.5% (w / v) IgePal, 50mM NaF, ImM Na3VO4, ImM dithiothreitol) containing ImM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (ThermoFisher). For analysis of whole colon, tissue was first sonicated in lysis buffer and protein concentration determined by DC protein assay kit (Bio Rad). For analysis of LP and lECs, cells were sonicated in RIPA buffer (150mMNaCl, 50mM Tris HC1 pH 7.5, 0.1% SDS, 1% Triton, 0.5% deoxyxholate) containing protease inhibitor cocktail (ThermoFisher). Samples were resolved by using SDS-PAGE and transferred to nitrocellulose membranes and analyzed by immunoblot with the indicated antibodies. Immunoreactivity was visualized by the Odyssey Imaging System (LICOR Biosciences).

[0119] Lamina propria and Intestinal epithelial cells extraction. For extraction of lECs, colon samples were added to Hanks’ Balanced Salt Solution (HBSS) containing 2.5mM EDTA, ImM DTT and 5% of fetal calf serum and incubated at 37°C with 300RPM for 30 minutes. Remaining colon pieces were then washed with PBS and further digested for LP cell extraction in HBSS containing 10% fecal calf serum, 0.5mg / ml collagenase I (Gibco), 0.5mg / ml collagenase IV (Millipore Sigma) and 0. Img / ml DNase I (Roche) at 37°C with 300RPM for 30 minutes. Samples were then passed through 1 0pm mesh and cells were collected by centrifugation at 900(xg) for 5 minutes. Cells were then subjected to MACS bead column-based separation. Cells were subjected to CD45 MACS MicroBeads (Mihtenyi Biotec) for CD45 positive LP cells and CD45 negative lECs. cDNA synthesis and real time quantitative PCR. Total RNA was extracted from whole colon and 1 pig of RNA was reverse transcribed using the iScript cDNA synthesis kit (Bio Rad). 5ng of cDNA was then subjected to qPCR analysis using iQ SYBR Green super-mix reagent (Bio Rad). Murine and human gene expression levels were normalized to TATA-binding protein (TBP) and GAPDH, respectively. Relative mRNA expression was calculated by a change in cycling threshold method as 2~ddC(t). Specificity of RT-qPCR amplification was assessed by melting curve analysis.

[0120] Colitis score. Colitis scoring was carried out blindly on freshly collected fecal samples using the following criteria: stool consistency: 0- well-formed pellets, 1- changed formed pellets, 2- loose stool, 3- diarrhea, occult: 0- no blood, 1- traces of blood, 2- moderate, 3- severe, 4- bleeding from anus.

[0121] Histology. Colon tissues were fixed in 10% neutral buffered formalin for 24- 48 hours before being processed and embedded in paraffin. Five micrometer thin sections were stained by H&E in an automated stainer (Leica Autostainer XL). Histomorphology of each H&E slide was evaluated by Applied Pathology Systems LLC at low and high-power field on an Olympus BX40 microscope, and the images were captured with Olympus cellSens Entry software at X4 magnifications. Grading of histology scores was performed blindly by Applied Pathology Systems LLC under the following scoring criteria. Inflammatory cell infiltrate was evaluated by the number of leukocyte foci: 0- no significant change, 1- mild, infiltrated leukocytes in focal or occasional, 2- moderate, infiltrated leukocytes with more than one focus and 3- severe, infiltrated leukocytes diffuse or continuous. Epithelial changes in goblet cell loss were evaluated by the reduction of goblet cell numbers relative to baseline goblet cell numbers per crypt: 0- no significant change, 1- goblet cell loss of 10% of normal amount, 2- goblet cell loss of 10-50% and 3- goblet cell loss higher than 50%. Crypt abscesses were evaluated by the neutrophils in crypt lumen: 0- no crypt abscess,

[0122] 1- rare crypt abscesses, 2- multiple crypt abscesses and 3- continuous crypt abscesses. Erosion was evaluated by the loss of surface epithelium: 0- no erosion, 1- one focus,

[0123] 2- multiple foci and 3- continuous surface loss. Hyperplasia was evaluated by the increase in crypt number or length relative to baseline, visible as crypt elongation or thickened crypt layers: 0- no hyperplasia, 1- mild, increase 50%, 2- moderate, increase of 50-100% and 3- marked, increase 100%. Ulceration was evaluated by crypt loss reaching beyond muscularis mucosae: 0- no ulceration, 1- one focus, 2- multiple foci and 3- continuous. Lymphoplasmacytic aggregates were evaluated by a score based on the location of lymphoplasmacytic aggregates reach: 0- no lymphoplasmacytic aggregates, 1- lymphoplasmacytic aggregates reach mucosa, 2- lymphoplasmacytic aggregates reach submucosa and 3- lymphoplasmacytic aggregates reach muscularis propria or subserosa.

[0124] Ki67 staining. Immunohistochemistry stain was performed by Applied Pathology Systems using detection kit (Vector Laboratories, MP-7601) on Dako autostainer. Paraffin tissues sectioned at 5 -micron thickness were dewaxed, rehydrated, and subjected to the antigen retrieval in citrate-based buffer pH 6.0 in a pressure cook. Slides were blocked with BloxAll blocking buffer and 2.5% Horse Serum respectively prior to 1 hour incubation with anti-Ki67 antibody at 1:250 dilution (abeam, abl 6667). Subsequently, the sections were incubated with anti-rabbit Amplifier antibody and ImmPress Excel polymer reagent sequentially before applying with DAB chromogen. The slides were then counterstained with hematoxylin followed by dehydration and coverslipping.

[0125] Flow cytometry. LP and IE cells were stained with anti-CD45.2 BV650, anti- CDllb BV510, anti-Ly6C APC, anti-CDl lc eF450, anti-CD64 BV711, anti-Ly6G PE-cy7, anti-Ly6G FITC, anti-CD3 PerCP-ef710, anti-B220 PE-Cy5, anti-CD326 APC. For mitochondrial mass and membrane potential, cells were stained with 120nM MitoTracker™ Green and MitoTracker™ Red (ThermoFisher), respectively. Cells were acquired on a CytekTM Aurora technology. Flow cytometry analysis was done with the FlowJo software. For sorting of colonic cells, cells were ectracted as deenbed under sterile conditions and sorted directly into complemented growing media. Sorting was performed on FACSAria III or FACSAria Fusion.

[0126] Antibiotic-induced microbiota depletion. 2-month-old WT mice were treated with autoclaved drinking water or autoclaved drinking water supplemented with 1 gr / L neomycin, Igr / L metronidazole, 0.5 gr / L vancomycin and 1 gr / L ampicillin for 1 month. Antibiotics were given individually or as a cocktail containing all 4 antibiotics. Fecal pellets were collected, and total DNA was extracted using DNeasy PowerSoil Kit (Qiagen) and subjected to qPCR using universal primers referred to as the 319F and 806R that hybridize to the V3-V4 region of the rRNA gene. Microbiome sequencing. Whole-genome shotgun (WGS) DNA sequencing libraries were constructed using Nextera XT DNA Library Prep Kits (Illumina Inc., San Diego, CA) and sequenced on a NextSeq500 Sequencing System as 150-base paired-end reads. Reads were trimmed reads and removed host decontamination using Tnmmomatic and Bowtie263, 64using Mus musculus genome assembly mmlO as the host reference. Reads were profiled for bacterial spices abundances using MetaPhlAn version 3.0.1 and database mpa_v292_CHOCOPhlAn_20190165.

[0127] Oxford Nanopore sequencing. Libraries for long-read Nanopore sequencing were generated with RNA from healthy distal colon. For mouse samples, RNA was enriched for poly(A) RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) and libraries were prepared using direct cDNA sequencing (SQK- DCS109) kits according to the manufacturer’s instructions. Due to limitation in RNA, libraries from human samples were prepared using cDNA-PCR sequencing (SOK- PCS109) kit. Libraries were sequenced with a MinlON IB device on an R9.4.1 flow cells (all Oxford Nanopore Technologies). Base calling was performed using guppy v4.2.2 (high accuracy) on Tesla V100 gpu (Nvidia). Full length reads were identified using pychopper v2.5.0 and filtered reads were aligned to the murine genome (assembly GRCm39 / mm39) or the human genome (assembly GRCh38 / hg38) using minimap2 v2.17 with the parameters ‘-ax splice -uf -k!4’. Annotations were generated using stringtie v2.1.5 with the parameters ‘-g 200 -L -conservative’ based on the aligned Nanopore reads and supported by Gencode annotation vM26 (mouse) or v38 (human). Aligned reads and annotations were visualized using IGV v2.9.4.

[0128] RNAseq analysis. Murine RNA sequencing dataset reported in this article can be found at the gene expression omnibus (ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE140788)24. RNA sequencing data was quantified with Salmon66(vl.9.0) using default parameters with respect to the concatenated GRCm39 cDNA and ncRNA transcriptome downloaded from ensemble67(release 109) without decoy sequences. Differential expression was computed with DESeq268(v3. 16) using the design formula (-batch + condition) in order to control for batch effects and genes with very low expression (rowCounts < 10) were pre-filtered. Volcano plots were plotted using R (EnhancedVolcano package, https: / / github.com / kevinblighe / EnhancedVolcano.) with IfcShrinkage using apeglm69, based on an adjusted p-value alpha of 0.05 and log2 fold-change threshold of 1. Heatmaps and clusters were generated using R (pheatmap package) with hierarchal clustering on row-normalized and DESeq2 rlog-normalized genes counts based on Euclidean distance (complete), after selecting for genes with an adjusted p-value<0.01 and |log2(FC)|>2.

[0129] Human IBD cohort dataset used in this study was the Mount Sinai Crohn’s and Colitis Registry (MSCCR) of biopsy whole transcriptome sequencing data of -1200 patients enrolled from December 2013-September 2016 and approved by the Icahn School of Medicine at Mount Sinai Institutional Review Board26, 27. Fastq files were trimmed using cutadapt using the options “-a AGATCGGAAGAGCACACGTCTGAACTCCAGTCA -minimum-length 1 -j 15”70(SEQ ID NO: 18). The trimmed fastq files were pseudoaligned and quantified using Salmon version 1.5.266. Salmon results files were imported into R using the package tximeta Bioconductor release 3.1471. Gene level counts were generated using the function summarizeToGene followed by TPM-normalization. The epithelial cell Seurat data object of single cell RNA-seq was retrieved from doi.org / 10.1016%2Fj. cell.2019.06.029 and subset to control and uninflamed samples. The percentage of epithelial cells with H0XA1 IAS expression greater than or equal to 1 TPM was compared to the total number of cells captured for each cell-type and the pre-existing epithelial cell identities were used.

[0130] In vitro translation assay. PcDNA3.1(+)-C-Myc vectors coding for HOXAllos potential ORFs were purchased from GenScript, USA. pCI-Caspasel vector was used as positive control72. For transient expression, 4X105HEK293T cells were transfected with 0.5 pg of the indicated vectors using lipofectamine™2000 (Invitrogen) as per manufacturer’s instructions. Cells were lysate in RIPA buffer supplemented with protease inhibitor cocktail 24 hours post infection. Samples were resolved on 12% SDS-PAGE and transferred to nitrocellulose membranes following analysis by immunoblot with anti Myc-Tag antibody (Cell signaling). Immunoreactivity was visualized by the Odyssey Imaging System (LICOR Biosciences).

[0131] In situ RNAscope analysis. Fresh colon biopsies were collected from the indicated mice and fixed in 4% PFA for 24 hours at 4°C. Samples were then immersed in 10% sucrose at 4°C for 24 hours, followed by 20% and 30% sucrose for additional 12-24 hours until the tissue sink to the bottom of the 15ml tube. Samples were then frozen in optimal cutting temperature (OCT) embedding media and store in tissue block container in -80°C until processing. Seven micrometer thin sections were mount on SuperFrost Plus slides and subjected to single-plex RNAscope 2.5 chromogenic assay BROWN and RNAscope multiplex fluorescent v2 assay according to Advanced Cell Diagnostic (ACD) user manual. Probes were purchased from ACD and are as followed:

[0132] Mm-Hoxal los-02: target region 575-1839bp, catalog number 1093151-C1 Mm-mt-Rnr2-C2: target region 25- 137bp, catalog number 590781 Positive control probes were included in the kits: Mm-Ppib-C2: targeting region 98-856 and Mm-Polr2a-Cl targeting region 34-860.

[0133] For single-plex RNAscope 2.5 chromogenic assay, slides were stained with 50% Hematoxylin staining solution and mounted using xylene-based mounting medium according to manufacturer’s instructions. Samples were visualized using TissueFAXS SL tissue cytometer by UMass Chan medical school SCOPE center (umassmed.edu / SCOPE / ).

[0134] For RNAscope multiplex fluorescent v2 assay, Mm-Hoxal los-Cl and Mm- mt-Rnr2-C2 were coupled with Opal™ 520 and Opal™ 690 Reagent Pack (1: 1500 dilution), respectively (Akoya bioscience). Samples were then stained with DAPI and mounted with ProLong Gold Antifade Mountant solution. Samples were visualized using Laser scanning confocal microscopy (Leica 8000) at 60x (oil) magnification. Images were taken with 0.2pm between z-slices in the differential interference contrast. For each biological replicate, at least 8 fields of view were imaged. IMARIS software (Bitplane) was used to analyze the colocalization in three dimensional images. Surface module was exploited to mark mitochondria and Spots module was used to mark HOXAllos. A filter with a negative threshold was used to identify HOXAllos within the mitochondria (beyond the mitochondrial surface). This data was analyzed blindly.

[0135] Subcellular fractionation. lECs and LP cells were isolated from the distal colon of WT mice and subjected to cell fractionation assay using RNA subcellular isolation kit (Active Motif). Briefly, 2x106cells were used for total RNA extraction and 4xl06cells for nuclear / cytoplasm extraction. Cell pellets were resuspended in complete lysis buffer and incubated for 20 minutes on ice. Lysates were centrifuge at 14,000g for 5 minutes at 4°C and supernatants were collected as the cytoplasmic fraction. Cell pellets, containing the nuclear fraction, were washed with 70% EtOH and centrifuge at 14,000g for 5 minutes at 4°C. Supernatants were removed, and Complete buffer G was added to each fraction. Samples were then subjected to column RNA extraction according to the protocol. RNA samples were treated with DNase I to remove genomic DNA and cDNA was synthesized and subjected to RT- qPCR. Expression levels of HOXA1 los, GAPDH and 7SK in each fraction were normalized to their expression levels in the input RNA, which was set as 100%.

[0136] Mitochondrial Isolation. Mitochondria were purified from colon biopsies collected from the distal colon of WT mice by Mitochondria Isolation kit MITOISO1 (Millipore Sigma). Briefly, mitochondria were isolated from colon biopsies by homogenization in Extraction Buffer A followed by centrifuges according to the manufacturer’s instructions. The final mitochondria fraction was resuspended in PBS containing 50ug / ml RNAse A for 20 minutes at room temperature. Mitochondrial RNA was extracted using Trizol.

[0137] ChIRP-MS. LP colonic cells freshly isolated from the distal colons were pooled from 18-23 mice (for each sample) and were subjected to ChIRP-MS analysis as previously described34. An amount of IxlO8cells were crosslinked using 3% FA for 30min at room temperature following by treatment with 2M glycine for lOmin at room temperature. Cells were then pelleted by spinning at 2000g for 3min at 4°C and lysis for sonication using water bath bioruptor in a 4°C at highest setting with 30sec on, 45sec off pulse intervals. Lysates were then spanned at 16,000g for lOmin at 4°C and supernatant was diluted in hybridization buffer. Ten percentages of the lysates were collected as input. Each sample was precleaned with C-l magnetic beads for 30min at 37°C. Samples were then collected and 1 pl of lOOpM HOXA1 los and control probe mix was added in a ratio of lul probe mix for each 1ml of sample. Following 4hr incubation at 37°C, C-l magnetic beads were added for 30min incubation at 37°C. Following, beads were washed 5 times and 10% of the resuspended beads were collected for RNA extraction using Trizol. Prior RNA extraction, beads were treated with proteinase K for 45min at 50°C followed by lOmin at 95°C. The remaining beads were used for protein extraction in 10% SDS. Samples were subjected to trypsin digestion followed by LC-MS / MS analysis at the UMass Chan Medical School MS core facility. RNA immunoprecipitation (RIP). RIP was performed using Magna RIP kit (Millipore Sigma) according to the manufacturer’s instructions on freshly isolated colonic LP cells isolated from H0XA1 los-KO and WT littermate control mice. Antibodies for NDUFV1 and mouse IgG control antibody were used.

[0138] Transmission Electron Microscopy. Fresh colon biopsies were collected from the indicated mice and immediately fixed in 2.5% glutaraldehyde / 1.6% paraformaldehyde in 0. 1 M Sodium Cacodylate buffer pH 7.2. and left overnight at 4°C. The samples were then rinsed three times in the same fixation buffer and postfixed with 1% osmium tetroxide for Ih at room temperature before rinsing three times with DH2O for 10 minutes, samples were then stained with 1% uranyl acetate for 30 mins at RT. and dehydrated through a graded ethanol series (10,30,50,70,85,95%), and 3x 100% ethanol. Samples were then infiltrated first with two changes of 100% Propylene Oxide before leaving overnight in a 50% / 50% propylene oxide I SPI-Pon 812 resin mixture. Over the following 2 days seven changes of fresh 100% SPI-Pon 812 resin were done before the samples were polymerized at 68°C in flat molds. The samples were then reoriented for horizontal sections of the colon tissue layers. The thin sections (approx. 70 nm) were placed on gold support grids, and contrasted with Lead citrate and Uranyl acetate. Sections were examined using the CM10 with 80Kv accelerating voltage, and images were captured using a Gatan TEM CCD camera.

[0139] Oxygen consumption rate (OCR). An amount of 75x105freshly colonic cells isolated from the distal colon were plated in XF96 cell culture plates in a non-CCh incubator. DMEM supplemented with ImM pyruvate, 2mM glutamine and lOmM glucose in a pH 7.4 was used. OCR was measured under basal conditions using the Seahorse XF extracellular flux analyzer (Agilent).

[0140] ATP measurements. An amount of 2xl06freshly lECs and LP cells isolated from the distal colon were lysate in ice cold lysis buffer containing lOOmM Tris, 2M NaCl, 20mM EDTA and 0.2% Triton X-100. Lysates were then centrifuge for lOmin at 16,000g and supernatants were collected for the measurement of ATP concentration by ATP determination kit (Molecular Probes A22066) and according to the manufacture’s protocol.

[0141] NAD+ / NADH measurements. An amount of 2xl04freshly lECs and LP cells isolated from the distal colon were lysate in 0.2N NaOH base solution containing 1% DTAB and subjected to NAD+ and NADH measurements by NAD / NADH-Glo™ Assay kit according to manufacturer’s protocol (Promega PRG9071).

[0142] Mitochondrial ROS measurements. An amount of 5X104freshly lECs and LP cells isolated from the distal colon were used for the detection of mictochondrial ROS by Mitochondrial ROS detection kit (Cayman chemical 701600). Antimycin A was used as positive control according to manufacturer’s protocol.

[0143] Mitochondrial DNA measurements. Real time PCR was performed on extracted DNA (QIAGEN) to determine mDNA / nDNA ratio. Primers targeting mitochondrial D-loop region were used to measure mDNA levels. Nuclear DNA levels were measured by primers targeting GAPDH.

[0144] Bone marrow chimera. Lethally irradiated (900R) 8-10-week-old recipient male mice were reconstituted by retro-orbital injection of 107bone marrow cells from age and gender matched donor mice. DSS experiments were performed 8 weeks post reconstitution.

[0145] Quantification and Statistical Analysis. Samples sizes were chosen with adequate statistical power based on previously published comparable studies. Differences between group means were tested with a t-test when the experiment contained two groups, or one-way ANOVA (followed by a Tukey post hoc test) when the experiment contained more than two groups. In cases where the experiment was running in two batches, “batch” was entered as a random categorical factor into a 2- way ANOVA. All pathology evaluation and takedowns were performed blindly. Data are presented as mean ± SEM. For in vivo and in vitro expenments, n represent number of animal or number of cells, respectively. Values of p < 0.05 were considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001.

[0146] For microbiome sequencing, ecological diversity metrics were generated using QIIME2 software version 2020.273. Beta diversity significance was tested using painvise PERMANOVA using Bray-Curtis dissimilarity. Differential abundance of taxa was evaluated using MaAsLin2 version 0.99.12 (Multivariate Analysis by Linear Models 2; huttenhower.sph.harvard.edu / maaslin2). Example 1. Murine IncRNA HOXAllos and the human ortholog HOXA11AS are uniquely expressed in the distal colon and downregulated during intestinal inflammation

[0147] To identify IncRNAs with regulatory function in UC, we performed transcriptome analysis of resident macrophages and infiltrated monocytes in the colon of mice that were either untreated or treated with dextran sodium sulfate (DSS) for 7 days to induce acute colitis24LncRNAs were differentially expressed in these cells under inflammatory conditions (Figure 1A-B). Immune genes (protein coding) in these cells were also differentially regulated, as expected. Given the large number of differentially expressed IncRNAs in the colon, we prioritized IncRNAs that were highly abundant in the colon, differentially expressed in a manner that correlates with colitis severity and conserved in humans. A IncRNA in the HOX locus, Homeobox Al 1 opposite strand (H0XA1 los) was amongst the most abundant IncRNAs in the healthy colon. HOXAl los was significantly downregulated under conditions of intestinal inflammation (Figure 1 A-B), suggesting a potential regulatory role for HOXAllos during intestinal homeostasis.

[0148] Unlike protein coding genes, IncRNAs are poorly annotated due to a lack of hallmark characteristics such as transcription initiation and termination. LncRNAs also undergo extensive alternative splicing, which increases the potential number of isoforms25. It is, therefore, essential to identify the H0XA1 los transcripts expressed in the colon. To this end, we utilized long read sequencing (Oxford Nanopore), a next generation sequencing method which allows for real time analysis of long RNA fragments and the identification of previously unannotated transcripts. Nanopore sequencing identified 4 independent transcripts of HOXAllos in the healthy colon of wild-type (WT C57BL / 6) mice (Figure 1C, Table 1). Transcripts 1 and 4 displayed high sequence similarity with minor differences in the second exon. Transcript 4 had a small number of splicing events relative to transcript 1 (Figure 1C), suggesting transcript 1 of H0XA1 los is more abundant. To quantify the expression levels of these isoforms, we used RT-qPCR with exon spanning primers to detect the mature / spliced form of each transcript (Figure ID). This analysis indicated that while transcript 3 was undetectable, transcript 2 was the most abundant transcript in the colon and this transcript was significantly downregulated in colitic mice (Figure ID). Thus, transcript 2, a novel isoform of H0XA11 os is abundant in the healthy colon. We next analysed a large human IBD cohort from the Mount Sinai Crohn’s and Colitis Registry (MDCCR) which included RNA-sequencing of colon biopsies from more than a thousand patients26, 27As in the mouse, the human ortholog, homeobox Al 1 antisense (H0XA1 IAS) was abundant in colon biopsies from healthy patients and significantly reduced in patients with UC (Figure IE). The levels of H0XA11 AS were decreased in a manner that correlated with disease severity (Figure IE). Interestingly, the levels of H0XA1 IAS were higher in adjacent non-inflamed areas of the colon in these patients (Figure IE).

[0149] We collected fresh colon biopsies from healthy human control patients and performed long read sequencing, which identified 2 transcripts of H0XA11 AS in the human colon (Figure 7A, Table 2, SEQ ID NOs:5 and 6). RT-qPCR using exon spanning primers showed that transcript 2 of H0XA1 IAS was the most abundant transcript in the healthy colon and this was reduced in the inflamed colon of patients with UC (Figure 7B). Murine H0XA1 los on chromosome 6 and human H0XA1 IAS on chromosome 7 are highly conserved IncRNAs with 80% identity as calculated by PFAAT (Figure 7C, Figure 12)28.

[0150] We next w anted to determine if there was any coding potential within the HOXAllos sequence. Through NCBI ORF finder, we identified 5 putative ORFs (Table 3). To test the coding potential of these putative ORFs, we cloned and transfected the myc-tagged ORFs into HEK293T cells. Western blotting revealed that the HOXAllos ORFs were not translated (Figure 7D). Thus, HOXAl los appears to lack coding potential.

[0151] Example 2. Murine HOXAllos and human HOXA11AS expression is restricted to the distal colon

[0152] LncRNAs are expressed in a more tissue restricted manner than protein coding genes, highlighting potential physiological functions in the tissues or cell types in which they are expressedlj 29. HOXA1 los expression was restricted to the distal colon of mice (Figure IF). In human tissue HOXA1 IAS demonstrated a similar expression pattern and was predominantly expressed in the distal colon when assessed across the intestinal tract (Figure 1G, Figure 7E)26‘27. HOXA1 los was expressed at high levels in CD45+ cells of the lamina propria (LP) and CD45' epithelial cells (lECs) (Figure 7F). To further assess the expression pattern of endogenous HOXAllos in colonic tissue, we utilized chromogenic RNA in situ hybridization (IHC), RNAscope, which enables the visualization of RNA at single molecule resolution in the intact colon (Figure 1H, Figure 7G). RNAscope analysis confirmed that H0XA1 los was highly abundant in the distal colon and undetectable in the proximal colon (Figure 1H). Within the distal colon, HOXAl los was expressed at high levels in lECs, LP and ly mphoid aggregates (Figure 7G). We also quantified HOXAllos expression in isolated cells from the distal colon. HOXAllos was expressed at high levels in both lECs and LP resident immune cells and was downregulated during colitis (Figure 7H). HOXAl los was undetectable in intraepithelial lymphocytes (lELs) under basal conditions (Figure 7H). These observations indicate that H0XA1 los is a conserved IncRNA expressed in both hematopoietic and non-hematopoietic cells of the distal colon.

[0153] Example 3. HOXAllos is localized to the mitochondria and interacts with mitochondria associated proteins

[0154] The functionality of IncRNAs depends on their sub-cellular localization30. Nuclear localized IncRNAs have been reported to regulate chromatin structure, transcription, RNA splicing, RNA stability and nuclear condensates. Whereas cytoplasmic localized IncRNAs impact mRNA stability, translation, protein localization and protein turnover. Cytoplasmic IncRNAs also regulate protein function by scaffolding of proteins operating in shared signaling pathways31, 32We therefore characterized the subcell ular localization of H0XA1 los in colonic LP cells (Figure 2A) or lECs (Figure 2B). As expected, the mature GAPDH mRNA transcript was enriched in cytosolic fractions (Figure 2A-B), while 7SK (a nuclear snRNA) was enriched in the nuclear fractions (Figure 2A-B). Mature H0XA1 los was enriched in the cytosolic fraction in both LP cells and lECs (Figure 2A-B).

[0155] We next generated mice lacking H0XA1 los through an innovative CRISPR / Cas9 approach that introduced a short 49-bp synthetic polyadenylation signal (spA)33in exon 1 proximal to the transcription start site of HOXAl los (Figure 8A). This resulted in a complete loss of HOXAl los transcripts 1 and 2 (Figure 8B) with no effect on the expression of the proximal protein coding gene H0XA11 under basal (Figure 8C-D) or colitis (Figure 8E-F) conditions.

[0156] We therefore sought to identify the protein binding partners of H0XA1 los. To this end, H0XA1 los-KO and littermate control mice were subjected to comprehensive identification of RNA-binding proteins by mass spectrometry (ChlRP- MS)34(Figure 2C). An amount of 1x108primary colonic LP cells were collected from the distal colon of 18-23 pooled H0XA11 os-KO or WT littermate control mice. The cells were then crosslinked and incubated with biotinylated antisense DNA probes designed to be complementary to the HOXAllos transcript and tiled along the full length of the transcript (Table 4). Probes targeting the Drosophila ROX gene were used as an irrelevant control. Probes were then captured by streptavidin magnetic beads followed by elution of RNA and proteins which were subsequently identified by liquid chromatography-mass spectrometry (LC-MS). Specific enrichment of endogenous H0XA1 los complexes using H0XA1 los specific probes was quantified by RT-qPCR. There was no enrichment of H0XA1 los when lysates from H0XA11 os-KO mice were incubated with specific probes, or when ROX probes were used. Further, GAPDH mRNA was undetectable following H0XA1 los pulldown, further confirming the specificity of HOXAllos antisense purification. We then subjected these pulldowns to mass spectrometry to identify H0XA11 os-associated proteins. Remarkably, H0XA1 los interacted with a number of enzymes with shared functions of metabolic pathways in the mitochondria (Figure 2C). Indeed, H0XA1 los interacted with mitochondrial proteins associated with the Krebs cycle, ETC and OXPHOS. H0XA1 los interacting proteins included a subunit of the mitochondrial membrane respiratory chain complex I (NADH dehydrogenase flavoprotein 1), proteins associated with fatty -acid oxidation and the Krebs cycle associated proteins Succinate coenzyme A (co A) ligase [ADP forming] subunit beta35, as well as Very long-chain specific acyl-CoA dehydrogenase (ACADVL), Isoform 2 of Acylcoenzyme A synthetase (ACSM3), Hydroxymethylglutaryl-CoA lyase (HMGCL), and Heat shock protein 75 kDa (TRAP1).

[0157] Recent studies have indicated that certain IncRNAs can localize to specific subcellular organelles30and regulate their activity36'38. Given that H0XA1 los interacts with proteins localized in the mitochondria, all of which are part of shared metabolic pathways, we next evaluated the sub-cellular localization of H0XA1 los. To this end, mitochondrial and cytoplasmic fractions w ere purified from distal colon tissue of WT mice and interrogated by qRT-PCR. As expected, the mRNA transcript of the mitochondrial encoded protein COXII was enriched in the mitochondrial fraction (Figure 2D), while the mRNA transcript of the nuclear encoded protein TBP (TATA-Binding Protein) was enriched in cytoplasmic fractions (Figure 2D). Mature HOXAllos was also enriched in the mitochondrial fraction (Figure 2D). These data were further confirmed by RNAscope (Figure 2E-H). HOXAllos and mitochondria were co-localized in biopsies collected from the distal colon of WT mice (Figure 2E). DAPI was used to stain cell nuclei. As a control, RNA polymerase 11 subunit A and Peptidylprolyl Isomerase B were also monitored (Figure 2F). Strikingly, we found that approximately 80% of mitochondria of the distal colon were highly enriched for HOXAllos (Figure 2G). No co-localization was observed between the control probes detecting RNA polymerase II subunit A (Figure 2G). HOXA1 los was also detected at low levels in the nucleolus (Figure 2E). The nuclear signal of HOXAllos likely represents newly transcribed, immature transcnpts of HOXA1 los. HOXA1 los was undetectable in the colon of HOXA1 los-KO mice underscoring the specificity of the probes used (Figure 2H).

[0158] Example 4. HOXA1 los-KO mice exhibit impaired OXPHOS and agedependent spontaneous intestinal inflammation

[0159] OXPHOS is a cellular process that harnesses the reduction of oxygen to generate phosphate bonds in the form of adenosine triphosphate (ATP) from adenosine diphosphate (ADP). Defects in OXPHOS have been associated with intestinal inflammation39'41. Given that HOXA1 los is localized to mitochondria and interacts with proteins coupled to mitochondrial OXPHOS, we next assessed whether deficiency in HOXAl los altered OXPHOS. Mitochondrial OXPHOS can be measured by monitoring the mitochondrial oxygen consumption rate (OCR). Colonic cells isolated from the distal colon of HOXA1 los-KO mice under basal conditions had lower OCR levels when compared to cells from WT littermate control mice (Figure 3 A), suggesting that HOXAllos deficiency results in a lower basal respiratory' rate.

[0160] HOXAllos deficient mice exhibited spontaneous intestinal inflammation as they aged, characterized by elevated levels of the pro-inflammatory cytokines IL-6 (Figure 3B), IL-10 (Figure 3C) and RANTES (Figure 3D) at 4-months of age when compared to WT littermate controls. HOXA1 los deficient mice displayed diarrhea and blood in their feces at 4-months of age. However, body weights and colon length were comparable between H0XA1 los-KO and their WT littermate controls. Histological features, immune cell composition and IEC composition were all comparable between HOXA1 los-KO and WT littermate controls. Example 5. HOXAllos deficient mice are highly susceptible to experimental models of colitis

[0161] Given that H0XA1 los deficiency predisposed animals to spontaneous inflammation and impaired OXPHOS in the colon, we hypothesized that H0XA1 los was protective for colitis. HOXAl los-KO and WT littermate control mice were subjected to acute colitis using dextran sulfate sodium (DSS) administered in the drinking water for 7 days (Figure 3E). H0XA1 los-KO mice were more susceptible to intestinal inflammation when compared to co-housed WT littermate controls (Figure 3F-Q), as demonstrated by shorter colon length (Figure 3F,J), increased weight loss (Figure 3G,K), higher score for stool appearance (Figure 3H-L) and higher pathological scores (Figure 3I,M-N). This enhanced susceptibility to colitis was observed with both male and female animals (Figure 3F-M). H0XA1 los-KO mice treated with DSS also had higher levels of pro-inflammatory cytokines (Figure 3O-Q) and Interferon stimulated genes (ISGs) when compared to WT littermate control mice. Thus, H0XA1 los deficiency sensitizes mice to colitis.

[0162] HOXAllos-KO mice also had a reduction in the level of the proliferative marker, Ki67, in the distal colon when compared to WT littermate control mice, suggesting a failure to restore barrier integrity (Figure 9A-B). We therefore subjected these mice to a recovery model of DSS-induced colitis to assess the role of H0XA11 os in the recovery phase of intestinal inflammation. H0XA11 os-KO and WT littermate control mice were treated with DSS for 5 days followed by a 7-day recovery period on regular water (Figure 9C). HOXAl los-KO mice continued to exhibit severe colitis during the recovery phase as demonstrated by shorter colon lengths (Figure 9D), diarrhea and traces of blood in the feces (Figure 9E), increased weight loss (Figure 9F) and higher pathological scores (Figure 9G-J). In contrast, littermate control mice recovered and had restored intestinal integrity (Figure 9D-J). In addition to DSS-induced colitis, HOXAl los-KO mice challenged with Salmonella enterica serovar Typhimurium SL1344 displayed shorter colon lengths (Figure 10A), greater weight loss (Figure 10B), higher colitis scoring (Figure IOC), higher pathological scores (Figure 10D-E) and elevated levels of the pro-inflammatory cytokines IL-6 (Figure 10F), ILl-lp (Figure 10G) and CXCL-10 (Figure 10H) when compared to co-housed WT littermate controls. We next characterized the contribution of the microbiome to the expression of HOXAllos in the distal colon using antibiotic-induced microbiota depletion (Figure 101). Following administration of a broad-spectrum antibiotic cocktail (containing neomycin, ampicillin, vancomycin and metronidazole) for 4 weeks which resulted in a complete depletion of the microbiota, WT mice expressed comparable levels of HOXAllos in the distal colon as those treated with regular water (Figure 101). Mice were also administrated individual antibiotics to deplete specific bacterial families. WT mice treated with neomycin (largely gram-negative targeting), ampicillin (broadspectrum targeting), vancomycin (largely gram-positive targeting) and metronidazole (largely anaerobic targeting) displayed similar expression levels of HOXA1 los as those treated with regular water (Figure 101). These data suggest that HOXA1 los expression is not dependent on the microbiota. We also assessed whether HOXAllos deficiency altered the composition of the microbiota in the colon, thus leading to increased susceptibility to acute colitis. Shotgun sequencing of stool samples collected from HOXA1 los-KO and WT littermate control mice demonstrated that HOXAllos deficiency had no effect on the colonialization of microbiota in the intestine under homeostatic conditions (Figure 10J).

[0163] Example 6. Deficiency of HOXAllos in myeloid cells promotes colitis

[0164] Given that H0XA1 los is expressed in both lECs and immune cells of the distal colon (Figure 7F-I), we next sought to understand the relative importance of each compartment in controlling intestinal homeostasis using bone marrow chimeras. Lethally Irradiated WT or HOXA1 los-KO mice were reconstituted with bone marrow from either WT or HOXA1 los-KO mice (Figure 4A). Eight weeks post reconstitution, mice were treated with DSS for 5 days followed by a 7-day recovery period on regular water. As expected, WT mice reconstituted with WT bone marrow cells (WT to WT) recovered from DSS treatment (Figure 4B-I). Similarly, HOXA1 los-KO mice reconstituted with WT bone marrow (WT to KO) completely recovered from DSS treatment (Figure 4B-I). Yet, WT mice reconstituted with KO bone marrow were hyper susceptible to colitis (Figure 4B-I). Thus, HOXAllos is important in hematopoietic cells to maintain gut homeostasis and restrict colitis. Example 7. HOXAllos deficient myeloid cells exhibits impaired Complex I activity

[0165] Fatty acids produced by the microbiota undergo fatty-acid oxidation to produce acetyl-CoA in colonocytes42, 43. Acetyl-CoA is then oxidized by the Krebs cycle coupled to the electron transport chain to produce ATP as part of OXPHOS. In this process, electrons fromNADH and FADH2, produced as part of the Krebs cycle, are transferred down the mitochondrial respiratory complexes to molecular oxygen. In this process, protons are pumped from the mitochondrial matrix to the intermembrane space and oxygen is reduced to form water. The energy released from these oxidation / reduction reactions is used to drive the synthesis of ATP from ADP. Defects in beta oxidation44-46, ETC and OXPHOS39-41have been associated with intestinal inflammation. Given that H0XA1 los deficiency in hematopoietic cells contributed to the increased susceptibility to colitis (Figure 4), we next assessed mitochondrial metabolic activity of LP CD45+immune cells (Figure 5A-C). We observed reduced NAD+ / NADH ratio (Figure 5A), suggesting impaired activity of the ETC complex I. Impaired conversion of NADH to NAD was coupled with a reduction in ATP production (Figure 5B). Inhibition of complex I leads to an increase in the production of mtROS due to the rapid generation of NADH47 48. Indeed, we observed elevated levels of mtROS in HOXAl los deficient immune cells (Figure 5C). HOXAl los deficient immune cells also showed reduced levels of mitochondrial DNA (mtDNA) when compared to WT littermate controls (Figure 5D). Reductions in the NAD+ / NADH ratio, ATP production and elevated levels of mtROS were also observed in lECs from H0XA1 los-KO mice. However, no alteration in mtDNA levels were found. Collectively, these data demonstrate that H0XA1 los regulates complex I activity in colonic myeloid cells and protects against mitochondrial damage.

[0166] Next, we performed RIP (RNA Immunoprecipitation) coupled to qPCR49to confirm that H0XA1 los interacts with the complex I subunit, NDUFV 1 which we had identified via ChiRP-MS (Figure 5E-F). H0XA1 los was highly enriched in the RNA fraction that immunoprecipitated with NDUFV1 (Figure 5E). H0XA1 los interaction with NDUFV 1 was specific, as we did not detect TBP in the NDUFV 1 pull downs or H0XA1 los in our IgG isotope controls (Figure 5E). In addition, HOXAllos was only detected in the input ofWT cells but not of H0XA1 los-KO cells, further demonstrating the specificity of the assay (Figure 5E). The specificity of the pull down was confirmed by western blotting (Figure 5F).

[0167] We next performed flow cytometry analysis of CD45+GDI lb+myeloid cells, CD45+GDI lb" lymphocytes and CD45 EPCM+lECs to measure mitochondrial mass and mitochondrial membrane potential (A'Pm) by using MitoTracker Green and MitoTracker Red, respectively50. We observed a significant decrease in both mitochondrial mass (Figure 5G-H) and A'Pm (Figure 5I-J) in H0XA1 los-KO myeloid cells. There was no further reduction of mitochondrial mass and A'Pm in HOXAllOs-KO myeloid cells treated with the complex I inhibitor, rotenone, indicating that complex 1 activity was already impaired in cells lacking H0XA1 los47(Figure 5G-I). Comparable levels of mitochondrial mass and A'Pm were observed in lymphocytes and lECs from H0XA1 los-KO and WT littermate control mice. These data support a role for H0XA1 los as a regulator of complex I activity and mitochondrial function in myeloid cells.

[0168] To provide additional evidence for the role of complex I deficiency in myeloid cells as the cause of inflammation in the colon of H0XA1 los deficient mice, we extracted CDllb+ myeloid cells from older mice and assessed the levels of pro- inflammatory cytokines. As expected, we observed higher levels of pro-inflammatory cytokines in in H0XA1 los-KO mice in comparison to their WT littermate counterparts (Figure 5K-M).

[0169] We then assessed the levels of mitochondrial markers and regulators in the H0XA1 los-KO immune cells (Figure 5N) and lECs by western blotting. We observed reduced levels of NDUFV1 in H0XA1 los-KO immune cells (Figure 5N) but not lECs. H0XA1 los-KO immune cells (Figure 5N) but not lECs also demonstrated reduced levels of the mitochondrial mass markers, citrate synthase and the mitochondrial import receptor subunit TOM20 as well as a reduction in the protein levels of the mitochondrial biogenesis markers, translocating-chain- associated protein (TRAM) and PPARG coactivator 1 alpha (PGCla). Mitochondrial fusion and fission play critical roles in maintaining functional mitochondria in response to environmental stress. Mitochondrial fusion serves to combine the contents of damaged mitochondria to healthy mitochondria whereas fission removes damaged mitochondria and facilitates mitophagy51. Indeed, levels of the fusion protein mitofusion 1 (MFN-1), the mitochondrial fission protein 1 (Fis-1) and the mitophagy regulator PARKIN were all elevated in H0XA1 los-KO immune cells (Figure 5N) but not lECs.

[0170] We then assessed mitochondrial morphology by transmission electron microscopy (TEM). We observed that mitochondria from HOXA1 los-KO myeloid cells were swollen with disrupted cristae and loss of matrix density when compared to intact mitochondrial ultrastructure in WT cells (Figure 50). Similar morphological changes have been previously reported by others in cells treated with rotenone52or cells with impaired A'Pm53. Interestingly, mitochondrial morphology in lECs from H0XA1 los-KO and WT littermate control mice demonstrated comparable, intact mitochondria. Taken together, these data demonstrate that H0XA1 los deficiency in colonic myeloid cells compromises mitochondrial function via dysregulated complex I.

[0171] To confirm that deficiency of complex I is responsible for the exacerbated colitis in H0XA1 los-KO mice, we treated mice with the complex I inhibitor rotenone. Rotenone was administrated locally via rectal administration to H0XA1 los-KO and WT littermate control mice every other day in the presence of 2% DSS (Figure 11 A). WT mice treated with rotenone were more susceptible to colitis to a degree similar to that observed in H0XA1 los-KO mice, as demonstrated by shorter colons (Figure 1 IB), greater weight loss (Figure 1 IB), traces of blood and diarrhea in the feces (Figure 1 ID), greater pathological score (Figure 11E-F) and elevated levels of pro-inflammatory cytokines (Figure 11G-I). Rotenone treatment of H0XA1 los-KO mice had no further impact on these mice. These data demonstrate that dysfunctional mitochondria as a result of complex I inhibition by rotenone or due to H0XA1 los-deficiency leads to enhanced susceptibility to colitis.

[0172] Example 8. Treatment with the mtROS scavenger, MitoQ, ameliorated colitis in HOXAllos-KO mice

[0173] Complex I deficiency leads to elevated mtROS in H0XA1 los-KO immune cells (Figure 5C). Excessive mtROS can cause oxidative stress, induction of inflammatory responses and activation of cell death pathways. MitoQ is a mitochondrial anti-oxidant that accumulates within the mitochondrial matrix to scavenge mtROS54To test if mtROS contributed to the exacerbated colitis phenotype observed in HOXAl los-KO mice, HOXAllos-KO and WT littermate control mice were treated with MitoQ every other day in the presence of 2% DSS for 7 days (Figure 6A). MitoQ ameliorated the enhanced colitis in H0XA1 los-KO mice treated with DSS. Indeed, MitoQ treated H0XA1 los-KO mice displayed comparable colon length (Figure 6B), weight loss (Figure 6C), blood and diarrhea (Figure 6D), pathological scores (Figure 6E-F, Figure 11 J) and pro-inflammatory cytokines (Figure 6G-I) as WT littermate control mice.

[0174] Collectively, these studies identify and characterize a IncRNA, H0XA1 los, that under basal conditions maintains complex I activity in colonic myeloid cells. In the absence of H0XA1 los, colonic myeloid cells have defective mitochondria, resulting in excessive production of mtROS and hyper susceptibility to colitis.

[0175] Table 1. Murine HOXAHos transcripts sequences.

[0176] Table 2. Human H0XA11 AS transcripts sequences

[0177] Table 3. Murine HOXAHos putative ORF sequences.

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[0263] OTHER EMBODIMENTS

[0264] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of diagnosing inflammatory bowel disease (IBD) in a subject, the method comprising: providing a sample from the subject; detecting a level of a H0XA11 AS in the sample; and comparing the level of the H0XA11AS in the sample to a reference level; wherein presence of a level of H0XA11AS below the reference level indicates that the subject has IBD.

2. A method comprising: providing a sample from the subject; and detecting a level of a H0XA11 AS in the sample; and optionally comparing the level of the H0XA11AS in the sample to a reference level.

3. A method of treating IBD in a subj ect, the method comprising: providing a sample from the subject; detecting a level of a H0XA11 AS in the sample; comparing the level of the H0XA11AS in the sample to a reference level; wherein presence of a level of H0XA11AS below the reference level indicates that the subject has IBD; and administering a treatment for IBD to the subj ect who has a level of HOXA 11 AS below the reference level.

4. The method of claims 1-3, wherein the sample comprises nucleic acids or cells from the intestine, optionally from the distal colon, of the subject.

5. The method of claim 4, wherein the sample comprises an intestinal biopsy or cytology brushing.

6. The method of any of claims 1-5, wherein the H0XA11AS is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:5 or 6.

7. The method of any of claims 1-6, wherein detecting a level of HOXA11AS comprises using RT-PCR.

8. A method of treating IBD in a subj ect, the method comprising administering to the subject a H0XA11 AS nucleic acid to cells in the intestine of the subject, e.g., to myeloid cells in the intestine of the subject.

9. The method of claim 8, wherein the H0XA11AS is at least 80%. 85%, 90%, 95%, or 99% identical to SEQ ID NO:5 or 6.

10. The method of claims 8 or 9, wherein the H0XA11 AS nucleic acid is naked RNA or a viral vector comprising a sequence encoding HOXA11AS.

11. An isolated nucleic acid encoding HOXA11AS, optionally wherein the HOXA11AS is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 5 or 6.

12. The isolated nucleic acid of claim 11, which is linked to or comprised within a nanoparticle, e.g., a lipid nanoparticle, optionally wherein the isolated nucleic acid is a HOXAHAS RNA.

13. An expression vector comprising a sequence encoding HOXA11AS operably linked to a promoter for expression of the HOXA11AS, optionally wherein the HOXA11AS is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 5 or 6.

14. The expression vector of claim 13, which is a viral vector, optionally an AAV, adenovirus, or lentivirus.

15. The method of claims 1 and 3-10, wherein the IBD is ulcerative colitis (UC).

16. AHOXA11AS nucleic acid for use in a method of treating IBD in a subject, the method comprising administering to the subject a H0XA11 AS nucleic acid to cells in the intestine of the subject, e.g., to myeloid cells in the intestine of the subject.

17. The HOXAllAS nucleic acid for the use of claim 16, wherein the HOXA1 IAS is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:5 or 6.

18. The H0XA11AS nucleic acid for the use of claims 16 or 17, wherein theH0XA11AS nucleic acid is naked RNA or a viral vector comprising a sequence encoding H0XA11AS.

19. The H0XA11 AS nucleic acid for the use of claims 6 or 17, wherein the H0XA11AS nucleic acid is in an expression vector, optionally wherein the expression vector is a viral vector, optionally an AAV, adenovirus, or lentivirus.

20. The HOXA11AS nucleic acid for the use of claims 16 to 19, wherein the IBD is ulcerative colitis (UC).

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