Genetically encoded systems for generating oxygen in living eukaryotic cells

Abstract

Described herein are compositions and methods for generating pericellular oxygen on surface of living eukaryotic cells, e.g., animal cells, by expressing a Cld enzyme (i.e., chlorite dismutase, chlorite O2-lyase, chlorite:O2 lyase), targeted to the plasma membrane of the cells.

Description

[0001] Attorney Docket No. 29539-0849WO1

[0002] GENETICALLY ENCODED SYSTEMS FOR GENERATING OXYGEN IN LIVING EUKARYOTIC CELLS

[0003] CLAIM OF PRIORITY

[0004] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 704,648, filed on October 8, 2024. The entire contents of the foregoing are incorporated herein by reference.

[0005] SEQUENCE LISTING

[0006] This application contains a Sequence Listing that has been submitted electronically as an XML file named “29539-0849WOl_SL_ST26.XML.” The XML file, created on October 7, 2025, is 66,670 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0007] TECHNICAL FIELD

[0008] Provided herein are compositions and methods for generating oxygen from chlorite on the surface of cells by targeting chlorite dismutase (Cid; also called chlorite O2-lyase and chlorite:O2 lyase) to the plasma membrane.

[0009] BACKGROUND

[0010] Oxygen is vital for all forms of life and is one of the most widely used substrates in all of biochemistry (Raymond and Segre 2006). One of the most important events for life on our planet was the great oxygenation event (GOE), some 2.1-2.4 billion years ago (Lyons 2014), which changed our environment and spawned aerobic life on our planet. Oxygen provides a thermodynamically favorable terminal electron acceptor that helps to power metabolism and has been proposed as a prerequisite for the emergence of complex forms of animal life (Nursall 1959). Since oxygen is a di-radical and can be toxic, numerous mechanisms evolved to allow organisms to safely wield its thermodynamic potential (Lu and Imlay 2021). In addition, oxygen plays a key role in signaling (Kaelin and Ratcliff 2018; Semenza 2012) and contributes to cell differentiation and development (Simon and Keith 2008). Humans have an absolute requirement for oxygen, only able to survive minutes in complete anoxia. At the other extreme, hyperoxia can also be devastating, leading to seizures, pulmonary toxicity, and retinopathy. Attorney Docket No. 29539-0849WO1

[0011] SUMMARY

[0012] Oxygen is fundamental to metabolism, signaling, and physiology, yet we lack robust genetic tools with which to manipulate its levels in living cells. Here we report the successful targeting of bacterial chlorite O2-lyase to the plasma membrane (PM- Cld), where it resides on the cell surface and catalyzes SupplemeNtal Oxygen Released from ChLorite (SNORCL). “Surface SNORCL” safely and rapidly generates large pericellular pulses of O2 that outpace its consumption by endogenous respiration. PM-Cld expression and activity are robust across multiple cell types and even in mouse xenograft tumors in vivo. We show that Surface SNORCL can be combined with the genetically encoded photosensitizer miniSOG2.0 - which generates toxic singlet O2 from O2 upon light activation - to elicit phototoxicity even under hypoxic conditions. We show that O2 generated by Surface SNORCL is sensed by the endogenous HIF system and influences signaling. Finally, we leverage the ability of PM-Cld to generate rapid O2 pulses to identify a set of 38 “fast-responding” O2-sensitive transcripts. PM-Cld expands the genetic toolkit for spatiotemporal control of O2, with anticipated applications in oxygen biology, medicine, and synthetic biology.

[0013] Accordingly, described herein are fusion proteins comprising: a bacterial or archaeal chlorite:O2 lyase (Cid), a secretory signal peptide, and a cell membrane tethering domain, wherein the secretory signal peptide directs the Cid to plasma membrane of a cell and the cell membrane tethering domain tethers the Cid enzyme to the plasma membrane.

[0014] In some embodiments, the Cid is from Nitrospira defluvii (M / Cld), Dechloromonas aromatica (ZL / Cld), or Nitrobacter winogradskyi (MrCld). In some embodiments, the Cid lacks a functional periplasmic targeting sequence. In some embodiments, the Cid comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the Cid is encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 36.

[0015] In some embodiments, the secretory signal peptide is positioned at the N- terminus of the Cid. In some embodiments, the secretory signal peptide is encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% Attorney Docket No. 29539-0849WO1 sequence identity to the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the secretory signal peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of any of SEQ ID NOs: 45-58.

[0016] In some embodiments, the cell membrane tethering domain is positioned at the C-terminus of the Cid. In some embodiments, the cell membrane tethering domain comprise a transmembrane domain. In some embodiments, the cell membrane tethering domain is encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 38; or wherein the cell membrane tethering domain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of any of SEQ ID NOs: 59-61.

[0017] In some embodiments, the Cid is directly fused to the secretory signal peptide and / or the cell membrane tethering domain.

[0018] In some embodiments, the fusion proteins further comprise one or more purification tags and / or linkers. In some embodiments, the one or more purification tags and / or linkers are positioned between the secretory signal peptide and the Cid. In some embodiments, the one or more purification tags and / or linkers are positioned between the Cid and the cell membrane tethering domain. In some embodiments, the fusion protein comprises one or more of HA tag, FLAG-tag, and 3xGGS linkers. In some embodiments, the one or more of HA tag, FLAG-tag, and 3xGGS linkers are positioned between the secretory signal peptide and the Cid. In some embodiments, the one or more of HA tag, FLAG-tag, and 3xGGS linkers are encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 35. In some embodiments, the fusion proteins comprise one or more Myc tag. In some embodiments, the one or more Myc tag is positioned between the Cid sequence and the cell membrane tethering domain. In some embodiments, the one or more Myc tag is encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 37.

[0019] In some embodiments, the fusion proteins comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 40, optionally omitting one or more Attorney Docket No. 29539-0849WO1 purification tags and / or linker sequences. For example, described herein are fusion proteins comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 40, but without one or more purification tags and / or linker sequences.

[0020] Also provided herein are nucleic acid molecules encoding the fusion proteins described hereinabove.

[0021] In some embodiments, the nucleic acid molecules comprise a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 39, optionally omitting nucleotide sequence encoding one or more purification tags and / or linker sequences. For example, described herein are nucleic acid molecules that comprise a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 39, but without nucleotide sequences encoding one or more purification tags and / or linker sequences.

[0022] In some embodiments, the nucleotide sequence is codon optimized for expression in a eukaryotic cell, optionally in an animal cell, e.g., a human cell.

[0023] Also provided herein are vectors comprising the nucleic acid molecules described hereinabove.

[0024] Also provided herein are host cells comprising the vectors described hereinabove. In some embodiments, the host cells express the fusion proteins described hereinabove. In some embodiments, the host cell is an animal cell, optionally a mammalian cell. For example, the host cell can be a human cell.

[0025] Also provided herein are isolated eukaryotic cells expressing the fusion proteins described hereinabove. In some embodiments, the Cid is expressed on the surface of the isolated eukaryotic cells. In some embodiments, the isolated eukaryotic cell is an animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell, optionally a CAR-T cell.

[0026] Also provided herein are methods for generating oxygen on the surface of eukaryotic cells by culturing the isolated eukaryotic cells described hereinabove in a medium comprising chlorite. In some embodiments, the medium comprises about 1 nM to about 500 pM chlorite. In some embodiments, the rate of oxygen generation is greater than the rate of oxygen utilization by respiratory chain of the cell. Attorney Docket No. 29539-0849WO1

[0027] Also provided herein are methods for enhancing and / or restoring toxicity of a genetic singlet oxygen (O21) generator in a cell, the method comprising expressing the genetic O21generator with the vectors described hereinabove in the cell.

[0028] Also provided herein are transgenic non-human uni- or multi-cellular eukaryotic organisms comprising the isolated eukaryotic cells described hereinabove. In some embodiments, the transgenic non-human uni- or multi-cellular eukaryotic organism is a worm, a plant, or a mouse.

[0029] Also provided herein are methods of generating oxygen in a transgenic non- human uni- or multi-cellular eukaryotic organism, the method comprising maintaining the organism described hereinabove in an environment comprising chlorite. In some embodiments, the environment comprises about 1 nM to about 500 pM chlorite.

[0030] Also provided herein are methods of generating oxygen on the surface of a eukaryotic cell, the method comprising culturing the host cell described hereinabove in a medium comprising chlorite. In some embodiments, the medium comprises about 1 nM to about 500 pM chlorite.

[0031] Also provided herein are isolated eukaryotic cells expressing (i.e., engineered to express) a bacterial or archaeal chlorite dismutase (Cid). In some embodiments, Cid is fused to a targeting sequence, optionally wherein the targeting sequence directs the Cid to the mitochondria or to the plasma membrane. In some embodiments, the Cid is expressed in the cytoplasm and the mitochondria. In some embodiments, the Cid enzyme is fused to a targeting sequence that directs the enzyme to surface of the cell where it is tethered to the plasma membrane and faces outside the cell. In some embodiments, the isolated cells also express (e.g., have been engineered to express) a chlorite transporter, e.g., an exogenous chlorite transporter which can be useful in cell types that don’t readily uptake chlorite. In some embodiments, the chlorite transporter is a sodium iodide symporter (NIS). In some embodiments, the NIS is encoded by SLC5A5, optionally comprising a sequence shown in Table 2. In some embodiments, the cells do not express a chlorite transporter, or do not express an exogenous chlorite transporter. In some embodiments, the isolated cells are animal cells, e.g., mammalian cells, e.g., human cells, optionally CAR-T cells.

[0032] In some embodiments, the bacterial chlorite dismutase (Cid) is from Nitrospira defluvii (NdCld), Dechloromonas aromatica (DaCld), or Nitrobacter Attorney Docket No. 29539-0849WO1 winogradskyi (NwCld). In some embodiments, the bacterial or archaeal chlorite dismutase (Cid) lacks a functional periplasmic targeting sequence.

[0033] Also provided herein are methods for generating oxygen in a eukaryotic cell, the method comprising culturing any of the cells described herein in a media comprising chlorite, e.g., 10 pm to 5 mM chlorite, or at least 10, 50, 70, 75, 100, 250, or 500 uM chlorite, or up to 1, 2.5, or 5 mM chlorite. In some embodiments, the chlorite is sodium chlorite. In some embodiments, the cell is viable in media comprising at least 1, 2.5, or 5 mM chlorite.

[0034] Also provided herein are transgenic non-human uni- or multi-cellular eukaryotic organism comprising a cell as described herein. In some embodiments, the organism is a worm or a mouse. Additionally provided are methods for generating oxygen in the transgenic non-human uni- or multi-cellular eukaryotic organisms, comprising maintaining the organism in an environment comprising chlorite,

[0035] In some embodiments, the chlorite is present at levels that would be toxic to a non-transgenic organism of the same species.

[0036] Additionally, provided herein are isolated Cid proteins that lack a functional periplasmic targeting sequence. In some embodiments, the Cid proteins comprise a sequence as disclosed herein, optionally without a tag (e.g., without FLAG) sequence. In some embodiments, a Cid protein is connected to targeting sequence to an organelle, such as the mitochondria. In some embodiments, a Cid protein is connected to targeting sequence to the surface of the cell, preferentially to the plasma membrane. Also provided are nucleic acids comprising a sequence encoding any of the isolated Cid proteins, and optionally a sequence encoding a sodium iodide symporter (NIS). In some embodiments, the NIS is encoded by SLC5A5. In some embodiments, one or both of the sequences are codon optimized for expression in a eukaryotic cell, e.g., an animal cell, e.g., a human cell. In some embodiments, the sequences encoding a Cid and a transporter, (e.g., NIS) are located on a single nucleic acid. In some embodiments, a ribosomal skip sequence can be used between the Cid and NIS. In some embodiments, the ribosome skip sequence is a “2A” skip sequence, e.g., T2A, a P2A, an E2A, or an F2A; see, e.g., Liu Z, et al. (2017) “Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector” Scientific Reports 7:2193. Also described herein are vectors comprising any of the Attorney Docket No. 29539-0849WO1 nucleic acids, optionally a bi-cistronic vector that encodes both the Cid and the NIS, for expression of both.

[0037] Further provided are host cells comprising the nucleic acids and / or vectors as described herein, and optionally expressing the Cid and / or NIS proteins. In some embodiments, the host cell is an animal cell, e.g., a mammalian cell, e.g., a human cell. In some embodiments, the bacterial chlorite dismutase (Cid) is from Nitrospira defluvii (NdCld), Dechloromonas aromatica (DaCld), or Nitrobacter winogradskyi (NwCld). In some embodiments, the bacterial chlorite dismutase (Cid) lacks a functional periplasmic targeting sequence. In some embodiments, the host cell also expresses a sodium iodide symporter (NIS). In some embodiments, the NIS is encoded by SLC5A5, optionally comprising a sequence shown in Table 1.

[0038] Additionally, provided herein are methods for generating oxygen in a eukaryotic cell, comprising culturing any one or more of the host cells described herein. In some embodiments, the culturing is in a media comprising 10 uM to 5 mM chlorite, or at least 50, 70, 75, 100, 250, or 500 pM chlorite, or up to 1, 2.5, or 5 mM chlorite.

[0039] 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.

[0040] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0041] DESCRIPTION OF DRAWINGS

[0042] FIGs. 1A-1C: Screening Cid variants for expression in human cells. (1A) Reaction catalyzed by the Cid enzymes. (IB) Structures of Cid enzymes from Nd (Lineage I) and Nw (Lineage II) (PDB accession # 3NN2 and 3QP1; N- and C-termini are represented with green and red spheres, respectively). (1C) Lysates from HeLa cells transduced with lentivirus for indicated constructs were subjected to SDS-PAGE Attorney Docket No. 29539-0849WO1 and immunoblotted to confirm expression of FL AG-tagged Cid variants, GFP, or loading control.

[0043] FIGs. 2A-2E: Cid expressed in human cells assembles properly with high activity. (2A) Size exclusion chromatography profile of purified Cld. (2B) SDS- PAGE analysis of purified M / Cld visualized with coomassie. (2C) Absorption spectra of purified M / Cld in the presence of 500uM ferricyanide with (reduced) or without (oxidized) 2.5mM dithionite. (2D) Time traces of molecular oxygen formation with different chlorite concentrations. (2E) Steady state kinetics of / Cld catalyzed oxygen production. Points represent average of four measurements and error bars show the standard error of the mean. Data were normalized to represent activity per 100,000 cells.

[0044] FIGs. 3A-3D: Three-day toxicity of sodium chlorite to human HeLa cells. HeLa cells expressing (3A) GFP, (3B) Cld, (3C) GFP+NIS, or (3D) / Cld+NIS were grown for three days with the indicated concentration of freshly prepared sodium chlorite. Cell counts and viability were assessed after 3 days of growth using a Vi-Cell BLU Cell Viability Analyzer. Shown is the mean + / - s.d. of triplicate measurements.

[0045] FIGs. 4A-4E: On-demand generation of oxygen using SupplemeNtal Oxygen Released from ChLorite (SNORCL) in human cells. The Agilent Seahorse XFe96 system was used to measure oxygen levels and oxygen consumption rates (OCR) in live, intact HeLa cells. (4A) Overview of the SNORCL system and reaction catalyzed by Cid and transport facilitated by NIS. (4B) Western blot of FLAG-M / Cld in HeLa cells. (4C) Seahorse intact cell oxygen consumption rate measurements at 1% ambient oxygen with sequential additions of piericidin+antimycin (1 pM each) and sodium chlorite (0 (black, circles), 1 mM (dark grey, triangles), or 5 mM (light grey, squares), as also shown in the key for FIG. 4D) in HeLa cells expressing GFP or M / Cld. (4D) Seahorse intact cell oxygen consumption rate measurements at 1% ambient oxygen with sequential additions of piericidin+antimycin (1 pM each) and sodium chlorite (0, 1 mM, 2.5 mM, or 5 mM) in HeLa cells expressing GFP + NIS or M / Cld + NIS. (4E) Traces of the average oxygen levels within two minutes upon sodium chlorite addition (black arrow) in the Seahorse experiments shown in FIGS. 4C-D. OCR: oxygen consumption rate; mean + / - s.e.m. of n = 4-6 biological replicates are shown in FIGs. 4C-4D. Attorney Docket No. 29539-0849WO1

[0046] FIGs. 5A-5D: Subcellular targeting of SNORCLs to generate oxygen in the cytosol or mitochondria. (5A) Overview of mitochondrial targeted SNORCL. (5B) Immunoblots of mitochondrial and cytosolic fractions expressing FLAG- Cld or mito-FLAG- / Cld. (5C) Seahorse intact cell oxygen consumption rate measurements at 1% ambient oxygen with sequential additions of pieri ci din+antimycin (1 mM each) and sodium chlorite (0, 0.5 mM, 1 mM, or 5 mM) in HeLa cells expressing FLAG-Af / Cld or mito-FLAG-Af / Cld, with or without NIS. (5D) Raw traces of the oxygen levels within two minutes upon sodium chlorite addition (black arrow) in the Seahorse experiments shown in Fig. 5c. OCR: oxygen consumption rate; mean + / - s.e.m. of n = 4-6 biological replicates are shown in Figs. 5c-5d.

[0047] FIGs. 6A-6C: (6A) Seahorse intact cell oxygen consumption rate measurements at 21% ambient oxygen with addition sodium chlorite (0, 1 mM, or 5 mM) in HeLa cells expressing GFP + NIS or Af / Cld + NIS. (6B) Seahorse intact cell oxygen consumption rate measurements at 21% ambient oxygen with sequential additions of piericidin+antimycin (1 pM each) and sodium chlorite (0, 1 mM, or 5 mM) in HeLa cells expressing GFP + NIS or Af / Cld + NIS. (6C) Cell counts by Hoechst 33432 staining immediately after the Seahorse experiments, performed approximately! hour after chlorite addition. Means ± standard deviations of n=4 samples are shown.

[0048] FIG. 7: HeLa cells expressing GFP (top left panel), Af / Cld (top right panel), GFP+NIS (bottom left panel), or Cld+NIS (bottom right panel) were treated with freshly prepared sodium chlorite for 30 minutes, washed, and measured viability four hours later, Cell counts and viability were assessed after 3 days of growth using a Vi-Cell BLU Cell Viability Analyzer. Shown is the mean + / - s.d. of triplicate measurements.

[0049] FIGs. 8A-8D: (8A) Immunoblot analysis of FLAG-Af / Cld in HeLa cells coexpressing NIS or mCherry. (8B) Seahorse permeabilized cell oxygen levels at 1% ambient oxygen with addition of sodium chlorite (0, 0.5 mM, 1 mM, or 5 mM) in HeLa cells expressing FLAG-Af / Cld + NIS or FLAG-Af / Cld + mCherry. (8C) Seahorse intact cell oxygen consumption rate measurements at 1% ambient oxygen with sequential additions of piericidin+antimycin (1 pM each) and sodium chlorite (0, 0.5 mM, 1 mM, or 5 mM) in HeLa cells expressing FLAG-Af / Cld + NIS Attorney Docket No. 29539-0849WO1 or FLAG- / Cld + mCherry. (8D) Traces of the oxygen levels within two minutes upon sodium chlorite addition (black arrow) in the Seahorse experiments shown in FIG. 8C. OCR: oxygen consumption rate; mean + / - s.e.m. of n = 4-6 biological replicates are shown in FIGs. 8B-8D.

[0050] FIGs. 9A-9Q: Plasma membrane (PM)-Cld localizes to the plasma membrane and exhibits high activity in many cell types. (9 A) Schematic and immunofluorescence of PM- and cyto-Cld in HEK293T cells co-stained with pan- cadherin and Hoechst. Scale bar, 50 pm. (9B-9D) Hypothetical structural models of PM-Cld embedded in the plasma membrane via the PDISPLAY™ transmembrane anchor, showing side, top-down, and bottom views. One of five Cid monomers (red) and heme groups (yellow) and membrane anchor (blue) are colored. (9E) Western blot of cell extracts and media from HEK293T cells expressing PM- or cyto-Cld ± NaClO?. (9F-9G) Time trace and kinetic analysis of O2 production in HEK293T cells expressing PM- or cyto-Cld, indicated concentrations correspond to NaClO? injection indicated by a solid arrow (mean ± SD, n = 3). (9H-9I) O2 traces and kinetics in A375 cells expressing PM- or cyto-Cld (n = 3). (9J-9L) O2 production in intact or permeabilized HEK293T, HeLa, and A375 cells after NaCICh injection. (9M) Western blot showing PM-Cld expression in NCI-HCC cells. (9N) O2 evolution in NCI-HCC suspensions following graded NaCICh addition. (90) In vivo tumor O2 traces after NaC102 or vehicle injection into WT or PM-Cld NCI-HCC xenografts, injection indicated by a solid arrow. (9P) O2 traces in HEK293T suspensions following drug addition (Antimycin A / Piericidin, Bam 15, or DMSO) and repeated NaC102 injection. (9Q) Intracellular O2 dynamics in HEK293T cells (MitoXpress Intra) following drug and NaC102 injection under 1% O2. Antimycin A was included unless otherwise noted.

[0051] FIGs. 10A-10D. Surface SNORCL re-sensitizes hypoxic cells to singlet oxygen generation. (10A) Schematic of PM-Cld control cells and co-expression of cytosolic or mitochondrial miniSOG2.0 (mSG). (10B) Representative confluency traces of HEK293T cells expressing PM-Cld with the indicated mSG variant, exposed to a single blue-light pulse at 21% O2 prior to the start of the experiment. (IOC) As in FIG. 10B, but cells were illuminated under acute hypoxia (1% O2) and then returned to 21% O2 for monitoring. (10D) As in FIG. IOC, but with NaCICh injected immediately prior to illumination. Attorney Docket No. 29539-0849WO1

[0052] FIGs. 11A-11B: PM-Cld is sensed by HIF and can be used to reveal transcripts “fast-responding” to O2. (HA) Western blot showing HIFla levels in HEK293T cells expressing PM-Cld or cyto-Cld under hypoxia, with treatments indicated. (11B) qRT-PCR traces of DDIT4 in PM- or cyto-Cld cells following NaCICh or Piericidin treatment (solid arrow).

[0053] FIGs. 12A-12B: PM-Cld localizes to the plasma membrane of A375 and Hela cells. (12A) Immunofluorescence localization of PM-Cld and cyto-Cld together with the plasma membrane surface marker Pan-Cadherin in fixed A375 cells. Cells were counterstained with Hoechst nuclear dye. Scale bar represents 50pm. (12B) Immunofluorescence localization of PM-Cld and cyto-Cld in HeLa cells.

[0054] FIG. 13: Quantitative conversion of ClOi’ to O2 by PM-Cld. Representative time trace of O2 production in HEK293T cells expressing PM-Cld. A bolus of substrate (amount indicated in figure legend) was injected into the cell suspension to induce an increase in O2 level.

[0055] FIG. 14: miniSOG2.0 expression is comparable in cytosol and mitochondria. Western blot showing extracts from HEK293T cells expressing cyto- Cld or PM-Cld and control, cytosolic miniSOG2.0 (cyto-mSG) or mitochondrial miniSOG2.0 (mito-mSG). Actin detected as loading control, Cid detected with Anti- NdCld antibody and miniSOG2.0 detected with an anti-HA tag antibody.

[0056] FIG. 15: qRT-PCR quantification of BNIP3L and Ca9 transcript reveals relative insensitivity to PM-Cld activation and Piericidin exposure under hypoxia. Line plots (akin to FIG. 11B) show the transcript behavior quantified by qRT-PCR of BNIP3L and Ca9 within the three-time: PM- and cyto-Cld treated with NaCICh, and PM-Cld treated with Piericidin A. Time courses are labeled on the respective line plots and describe the localization of the SNORCL tool as well as the perturbation (NaCICh or Piericidin).

[0057] DETAILED DESCRIPTION

[0058] Oxygen (O2) is central to life. It is used in hundreds of biochemical reactions and serves as a thermodynamically favorable terminal electron acceptor for powering aerobic metabolism. O2 is also central to cellular signaling, developmental patterning, and systemic physiology. Despite the importance of O2, manipulating its levels with fine spatiotemporal resolution remains a challenge. Currently, to modulate O2 levels, Attorney Docket No. 29539-0849WO1 cells or animals are typically placed in airtight chambers into which varying gas mixtures can be introduced. Not only does this approach require specialized equipment (e.g., hypoxia chambers, gas cylinders, regulators), but because oxygen is poorly soluble in water, it also takes a long time for the gas to diffuse into and across biofluids. In the context of entire organisms, because of systemic physiology and homeostasis, it is not possible to manipulate the O2 levels at a particular organ or cell type of interest with specificity.

[0059] In recent years, genetically encoded tools have been developed and widely adopted for generating activated forms of O2. These tools include D-AAO (Steinhorn et al., 2018), KillerRed (Bulina et al., 2006), and miniSOG (Shu et al., 2011), which produce H2O2, superoxide, or singlet oxygen CO2), respectively, with spatiotemporal precision. However, such genetic tools have been largely lacking for generating molecular O2 itself. We previously introduced SupplemeNtal Oxygen Released from ChLorite (SNORCL), a genetically encoded O2-producing system based on the bacterial enzyme family chlorite:O2 lyase (Cid) (Markhard et al., 2022). Compositions and methods for generating oxygen in living eukaryotic cells, e.g., animal cells, by expressing chlorite dismutase (Cid; also called chlorite O2-lyase and chlorite:O2 lyase) in the cells are described in WO2023 / 211896 (PCT / US2023 / 019755), the contents of which are incorporated herein by reference in its entirety. We chose to focus on the Cid family of oxidoreductases as a chassis for a simple-to-use oxygen generator given that its substrate is bioorthogonal to eukaryotic metabolism. In WO2023 / 211896 (PCT / US2023 / 019755), we have shown that when expressed in human cells, Cid enzymes exhibit high activity, and that we can coexpress plasma membrane transporters that promote uptake of sodium chlorite for its subsequent intracellular conversion to oxygen. In this way, we were able to successfully deploy a genetic system for SupplemeNtal Oxygen Released from ChLorite (“SNORCL”; also sometimes called SupplemeNtal Oxygen via Reduction of ChLorite).

[0060] Cid enzymes catalyze O2 formation from chlorite (CIO2 ) via a non-covalently bound, distal histidine-coordinated heme b cofactor, representing one of the few biological mechanisms known to form 0=0 bonds (Lee et al., 2008). These enzymes exhibit diverse substrate affinities and catalytic efficiencies across bacterial species, with Nitrospira defluvii Cid (M / Cld) showing a catalytic turnover rate (kcat) of 82 s ' Attorney Docket No. 29539-0849WO1 at neutral pH (Schaffner et al., 2015). In our prior work, we provided proof of concept that M / Cld, when expressed either in the cytosol or targeted to the mitochondrial matrix of mammalian cells, could yield localized O2 production. Such cytosol expressed Cid is referred to herein as cyto-Cld. However, transmembrane import of CIO2 was poor with cyto-Cld, and increasing substrate concentrations was precluded by its toxicity. Although we could improve substrate import with a coexpressed human NIS-transporter (SLC5A5 rates of O2 remained modest relative to endogenous rates of its consumption.

[0061] Here, we sought to overcome this substrate import limitation by tethering / Cld to the exterior-facing side of the plasma membrane, thus generating plasma membrane Cid (PM-Cld), enabling immediate conversion of externally added CIO2 into freely diffusible O2 (FIG. 9A). What was unclear was whether membrane trafficking and anchoring through the ER and Golgi might impair proper heme incorporation, folding, and activity of the enzyme. However, given the existence of several membrane-associated and secreted heme-containing enzymes (Shao & Hegde, 2011; Noreng et al., 2022), we hypothesized that an outward-facing PM-Cld would similarly retain catalytic activity.

[0062] Accordingly, described herein are plasma membrane targeted Cid enzymes. Plasma membrane targeted Cid enzymes (e.g., M / Cld enzymes) of the present disclosure can be referred to herein as PM-Cld or PM-M / Cld. PM-Cld can reside on the cell surface and catalyze SNORCL, thus generating oxygen on cell surface. Oxygen generated on cell surface is produced exteriorly and is taken up by the cells across the cell membrane. Oxygen generated on surface of cells can be referred to herein as pericellular oxygen. Thus, described herein is “Surface SNORCL”, a system that targets Cid to the surface of cells, i.e., to the plasma membrane of cells, whereupon oxygen is produced exteriorly and is taken up by the cells across the cell membrane. The present disclosure shows that Surface SNORCL can safely and rapidly generate large pericellular pulses of oxygen that outpace its consumption by endogenous respiration. PM-Cld expression and activity are robust across multiple cell types and even in mouse xenograft tumors in vivo. Also, PM-Cld can convert even non-toxic doses of chlorite to generate oxygen (e.g., pericellular oxygen) at the surface of cells. The present disclosure also shows that Surface SNORCL can be combined with the genetically encoded photosensitizer miniSOG2.0 - which Attorney Docket No. 29539-0849WO1 generates toxic singlet oxygen from oxygen upon light activation - to elicit phototoxicity even under hypoxic conditions. Furthermore, the present disclosure shows that oxygen (e.g., pericellular oxygen) generated by Surface SNORCL is sensed by the endogenous HIF system and influences signaling. Also, the present disclosure shows the ability of PM-Cld to generate rapid oxygen (e.g., pericellular oxygen) pulses to identify a set of 38 “fast-responding” oxygen-sensitive transcripts. PM-Cld expands the genetic toolkit for spatiotemporal control of oxygen, with anticipated applications in oxygen biology, medicine, and synthetic biology.

[0063] Accordingly, described herein are PM-Cld, and use and expression thereof in eukaryotic cells.

[0064] PM-Cld

[0065] Described herein are Cid enzymes (e.g., / Cld) that are targeted to plasma membrane of cells (e.g., mammalian cells, such as human cells). Plasma membrane targeted Cid enzymes (e.g., / Cld enzymes) of the present disclosure can be referred to herein as PM-Cld or PM-Cld fusion protein. For example, plasma membrane targeted / Cld enzymes can be referred to herein as PM- / Cld or PM- / Cld fusion protein. Also described herein are systems and methods for targeting Cid enzymes to plasma membrane of cells, e.g., for generation of PM-Cld. Such systems and methods can be referred to herein as Surface SNORCL or Surface-SNORCL. Surface SNORCL can use constructs that target Cid to the surface of cells, i.e., to the plasma membrane of cells, whereupon oxygen can be produced exteriorly and taken up by the cells across the cell membrane. Constructs for use with Surface SNORCL can include, e.g., a Cid enzyme (e.g., any of the Cid enzymes described herein) fusion protein that is targeted to the plasma membrane of cells (e.g., via one or more of the secretory signal peptides described herein) wherein the proteins are tethered to the plasma membrane (e.g., via one or more of the cell membrane tethering proteins described herein). These constructs can be generated, for example, by placing a nucleotide sequence encoding the Cid in a vector that encodes the fusion proteins that are targeted to the plasma membrane of cells (e.g., via one or more of the secretory signal peptides described herein) and tethered to the plasma membrane (e.g., via one or more of the cell membrane tethering proteins described herein). For example, Attorney Docket No. 29539-0849WO1

[0066] Surface SNORCL can be generated by placing a nucleotide sequence encoding Cid (e.g., / Cld) enzymes in a pDisplay™ system.

[0067] PM-Cld of the present disclosure can be a fusion protein comprising a Cid enzyme, a secretory signal peptide (e.g., for targeting the Cid enzyme to the plasma membrane), and a cell membrane tethering domain (e.g., for tethering the Cid enzyme to the plasma membrane). The Cid enzyme can be fused directly to the secretory signal peptide and / or the cell membrane tethering domain. Alternatively, the Cid enzyme can be fused to the secretory signal peptide and / or the cell membrane tethering domain via one or more linkers, purification tags, and / or other intervening sequences. The secretory signal peptide can be positioned at the N-terminus of the Cid enzyme. Alternatively, the secretory signal peptide can be positioned at the C- terminus of the Cid enzyme. The cell membrane tethering domain can be positioned at the C-terminus of the Cid enzyme. Alternatively, the cell membrane tethering domain can be positioned at the N-terminus of the Cid enzyme. For example, from the N-terminus to the C-terminus, PM-Cld fusion proteins can comprise a secretory signal peptide, a Cid enzyme, and a cell membrane tethering domain.

[0068] PM-Cld fusion proteins of the present disclosure can comprise an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 40, optionally omitting one or more purification tags and / or linker sequences. For example, PM-Cld fusion proteins can comprise an amino acid sequence having the amino acid sequence of SEQ ID NO: 40, optionally omitting one or more purification tags and / or linker sequences. Additionally, or in the alternative, PM-Cld fusion proteins of the present disclosure can be encoded by a nucleotide sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleotide sequence of SEQ ID NO: 39, optionally omitting nucleotide sequences encoding one or more purification tags and / or linker sequences. For example, PM-Cld fusion proteins can be encoded by the nucleotide sequence of SEQ ID NO: 39, optionally omitting nucleotide sequences encoding one or more purification tags and / or linker sequences. Attorney Docket No. 29539-0849WO1

[0069] Cid enzyme

[0070] PM-Cld of the present disclosure can comprise any of the Cid enzymes described herein.

[0071] Chlorite dismustases (Cid) are heme b-containing oxidoreductases that are found in bacteria including Proteobacteria, Cyanobacteria, and Nitrospirae, as well as in archaea. Cid useful in the present methods and compositions have chlorite decomposition activity; an exemplary Cid is homo-pentameric (Lineage I, e.g., from Dechloromonas aromatica (DaCld) and Nitrospira defluvii ( / Cld)) or homo-dimeric (Lineage II, e.g., from Nitrobacter winogradskyi (NwCld)). See, e.g., Hofbauer et al., Biotechnol J. 2014 Apr, 9(4): 461-473; Kostan et al., J. Struct. Biol. 2010, 172:331— 342; van Ginkel, Arch Microbiol. 1996 Nov, 166(5):321-6; Goblirsch, B. et al. J Mol Biol 408(3):379-98 (2011); Coates and Achenbach, Nat Rev Micro 2, 569-580 (2004); and US 10724010. Exemplary sequences are known in the art and include those provided herein (optionally lacking the FLAG (DYKDDDDK (SEQ ID NO: 1)) sequence and any linkers, e.g., GS-rich linkers (GGSGGSGGS (SEQ ID NO:2))) as well as those in the preceding references, particularly those disclosed in Table 1 of US10724010, including RefSeq accession numbers YP_005026408.1, YP_285781.1, AAM92878.1, WP_014235269.1, AAT07043.1, WP_009867516.1, CAC14884.1, WP_013516316.1, ACA21503.1, YP_004267835.1, EFH80711.1, YP_004178041.1, YP_004367213.1, YP_004058724.1, or YP_004172359.1. In preferred embodiments, the sequences useful herein have an arginine residue at the distal side of heme b (Hofbauer et al., Biotechnol J. 2014 Apr, 9(4): 461-473) required for chlorite degradation. The sequences can lack a periplasmic targeting sequence, and are preferably codon optimized for expression in a host cell. The sequences can include a signal targeting them to a specific subcellular compartment, e.g., a mitochondrial targeting presequence and / or internal signal, see, e.g., Truscott et al., Current Biology, Vol. 13, R326-R337, April 15, 2003. Exemplary Cid enzymes are shown in Table 1.

[0072] TABLE 1. Cid enzymes

[0073] An exemplary sequence of / Cld lacking a periplasmic targeting sequence is: Attorney Docket No. 29539-0849WO1

[0074] MADREKLLTESGVYGTFATFQMDHDWWDLPGESRVISVAEVKGLVEQWSG KILVESYLLRGLSDHADLMFRVHARTLSDTQQFLSAFMGTRLGRHLTSGGLLHGVSK KPTYVAGFPESMKTELQVNGESGSRPYAIVIPIKKDAEWWALDQEARTALMQEHTQ AALPYLKTVKRKLYHSTGLDDVDFITYFETERLEDFHNLVRALQQVKEFRHNRRFGH PTLLGTMSPLDEILEKFAQ (SEQ ID NO: 3) .

[0075] A useful sequence to target any of the Cid proteins described herein to the mitochondria comprises:

[0076] MLATRVFSLVGKRAISTSVCVRAH (SEQ ID NO:4).

[0077] Transporters that promote uptake of chlorite include human sodium iodide symporter (NIS), encoded by solute carrier family 5 member 5 (SLC5A5), and homologs thereof, e.g., as shown in Table 2. Described herein are cells (e.g., eukaryotic cells) expressing PM-Cld fusion proteins of the present disclosure, wherein the cells do not express a chlorite transporter, or do not express an exogenous chlorite transporter.

[0078] TABLE 2. Chlorite transporters

[0079] PM-Cld can comprise an Af / Cld enzyme. The AWCld enzyme can lack a periplasmic targeting sequence. For example, PM-Cld of the present disclosure can comprise an Af / Cld enzyme that comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 3; such as, an Cld enzyme that comprises the amino acid sequence of SEQ ID NO: 3. Additionally, or in the alternative, PM-Cld of the present disclosure can comprise an At / Cld enzyme that is encoded by a nucleotide sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) Attorney Docket No. 29539-0849WO1 sequence identity to the nucleotide sequence of SEQ ID NO: 36. For example, PM- Cld of the present disclosure can comprise an / Cld enzyme that is encoded by the nucleotide sequence of SEQ ID NO: 36.

[0080] Secretory signal peptide

[0081] PM-Cld of the present disclosure can comprise a secretory signal peptide. A secretory signal peptide can also be referred to herein as a secretion signal, a secretory signal peptide sequence, or a targeting sequence. The secretory signal peptide can target (i.e., direct) the Cid to the surface (e.g., plasma membrane) of a cell and expose the Cid to the exterior of the cell. The Cid enzyme can be fused directly to the secretory signal peptide. Alternatively, the Cid enzyme can be fused to the secretory signal peptide via one or more linkers, purification tags, and / or other intervening sequences.

[0082] A secretory signal peptide for use in a PM-Cld fusion protein of the present disclosure can be a IgK secretion signal. The IgK secretion signal can be encoded by a nucleotide sequence that has at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleotide sequence of SEQ ID NO: 34. For example, the IgK secretion signal can be encoded by the nucleotide sequence of SEQ ID NO: 34.

[0083] A secretory signal peptide for use in a PM-Cld fusion protein of the present disclosure can be a secretory signal peptide known in the art. A number of secretory signal peptides, including human secretory signal peptides, are described in the art, e.g., in Table 5 of US10993967; von Heijne (J Mol Biol. 1985 Jul 5, 184(l):99-105); Kober et al. (Biotechnol. Bioeng. 2013, 110: 1164-1173); and Tsuchiya et al. (Nucleic Acids Research Supplement No. 3 261 -262 (2003)). Examples of human secretory signal peptides are shown in Table 3 (Table is adapted from novoprolabs.com / support / articles / commonly-used-leader-peptide-sequences- forefficient- secretion-of-a-recombinant-protein-expressed-in-mammalian-cells- 201804211337.html). Thus, a secretory signal peptide for use in a PM-Cld fusion protein of the present disclosure can comprise an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, Attorney Docket No. 29539-0849WO1

[0084] 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of any of SEQ ID NOs: 45-58.

[0085] TABLE 3. Exemplary Human Secretory Signal Peptide Sequences

[0086] Cell membrane tethering domain

[0087] PM-Cld of the present disclosure can comprise a cell membrane tethering domain. A cell membrane tethering domain can also be referred to herein as a cell membrane tethering domain sequence or a plasma membrane tethering domain. The cell membrane tethering domain can tether (i.e., anchor) the Cid enzyme to the plasma membrane of a cell. For example, the cell membrane tethering domain can tether the Cid enzyme to the plasma membrane of a cell, thus preventing secretion of the Cid enzyme. The Cid enzyme can be fused directly to the cell membrane tethering domain. Alternatively, the Cid enzyme can be fused to the cell membrane tethering domain via one or more linkers, purification tags, and / or other intervening sequences. The cell membrane tethering domain can comprise a transmembrane-intracellular domain or a transmembrane domain. The cell membrane tethering domain can comprise a cell surface receptor, or a cell membrane-bound portion thereof.

[0088] A cell membrane tethering domain for use in a PM-Cld fusion protein of the present disclosure can be a transmembrane domain. For example, a cell membrane tethering domain for use in a PM-Cld fusion protein of the present disclosure can be a Attorney Docket No. 29539-0849WO1

[0089] PDGFR transmembrane domain. The PDGFR transmembrane domain can be encoded by a nucleotide sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleotide sequence of SEQ ID NO: 38. For example, the PDGFR transmembrane domain can be encoded by the nucleotide sequence of SEQ ID NO: 38.

[0090] A cell membrane tethering domain for use in a PM-Cld fusion protein of the present disclosure can be a transmembrane domain from transferrin receptor protein. For example, a cell membrane tethering domain for use in a PM-Cld fusion protein of the present disclosure can be a transmembrane domain from human transferrin receptor protein 1 (TFR1). Exemplary sequence of human TFR1 is provided at UNIPROT Accession No. P02786. The transmembrane domain of human TFR1 can comprise amino acids 68-88 of TFR1. The TFR1 transmembrane domain can comprise an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 59. For example, the TFR1 transmembrane domain can comprise the amino acid sequence of SEQ ID NO: 59. A cell membrane tethering domain for use in a PM-Cld fusion protein of the present disclosure can be a transmembrane domain from human transferrin receptor protein 2 (TFR2). Exemplary sequence of human TFR2 is provided at UNIPROT Accession No. Q9UP52. The transmembrane domain of human TRF2 can comprise amino acids 84-104 of TFR2. The TFR2 transmembrane domain can comprise an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 60. For example, the TFR2 transmembrane domain can comprise the amino acid sequence of SEQ ID NO: 60.

[0091] A cell membrane tethering domain for use in a PM-Cld fusion protein of the present disclosure can be a transmembrane domain from HLA class II histocompatibility antigen gamma chain (HG2A). For example, a cell membrane tethering domain for use in a PM-Cld fusion protein of the present disclosure can be a transmembrane domain from human HG2A (encoded by CD74 gene). Exemplary sequence of human HG2A is provided at UNIPROT Accession No. P04233. The Attorney Docket No. 29539-0849WO1 transmembrane domain of human HG2A can comprise amino acids 47-72 of HG2A. The HG2A transmembrane domain can comprise an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 61. For example, the HG2A transmembrane domain can comprise the amino acid sequence of SEQ ID NO: 61.

[0092] A cell membrane tethering domain for use in a PM-Cld fusion protein of the present disclosure can be a cell membrane tethering domain known in the art. For example, a cell membrane tethering domain for use in a PM-Cld fusion protein of the present disclosure can be a transmembrane domain known in the art. A number of transmembrane domains are described in the art, e.g., in US Patent No. US10993967; and US Patent Application Publication No. US20230051406.

[0093] Purification tags and linkers

[0094] PM-Cld of the present disclosure can also comprise one or more purification tags and / or linkers. The purification tags and / or linkers can be positioned between the secretory signal peptide and the Cid. The purification tags and / or linkers can also be positioned between the Cid and the cell membrane tethering domain.

[0095] Purification tags for use in PM-Cld of the present disclosure can be one or more of HA tag, FLAG tag, Myc tag (EQKLISEEDL; SEQ ID NO: 41), His tag, and biotin tag. For example, PM-Cld of the present disclosure can comprise one or more of HA tag, FLAG-tag, and 3xGGS linkers that are encoded by a nucleotide sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleotide sequence of SEQ ID NO: 35. The one or more of HA tag, FLAG- tag, and 3xGGS linkers can be positioned between the secretory signal peptide and the Cid. Alternatively, or in addition, the one or more of HA tag, FLAG-tag, and 3xGGS linkers can be positioned between the Cid and the cell membrane tethering domain. PM-Cld of the present disclosure can also comprise one or more Myc tag encoded by a nucleotide sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleotide sequence of SEQ ID NO: 37. The one or more Myc tag can be positioned between the Cid sequence and the cell membrane Attorney Docket No. 29539-0849WO1 tethering domain. Alternatively, or in addition, the one or more Myc tag can be positioned between the secretory signal peptide and the Cid.

[0096] Linkers for use in PM-Cld fusion proteins of the present disclosure can be linkers that are described, for example, in Argos, Mol Biol 211 : 943-958, 1990; George and Heringa, Protein Eng 15:871-879, 2002; Chen et al., Biotechniques 49:513-518, 2010; and Chen et al., Adv Drug Deliv Rev 65(10): 1357-1369, 2013.

[0097] A linker for use in a PM-Cld fusion protein described herein can be a flexible linker, a rigid linker, and / or an in vivo cleavable linker. For example, a linker for use in PM-Cld fusion protein can be a flexible linker. Flexible linkers are usually applied when the protein domains that need to be joined require a certain degree of movement or interaction. They are generally composed of small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The small size of these amino acids provides flexibility, and allows for mobility of the connecting functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules, and therefore reduce the unfavorable interaction between the linker and the protein moieties. An example of the most widely used flexible linker is the sequence (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: 42).

[0098] Additionally, or in the alternative, a linker for use in PM-Cld fusion proteins described herein can be a rigid linker. While flexible linkers have the advantage of connecting the functional domains passively and permitting a certain degree of movement, the lack of rigidity of these linkers can be a limitation. There are several examples in the literature where the use of flexible linkers resulted in poor expression yields or loss of biological activity. Under such situations, rigid linkers can be successfully applied to keep a fixed distance between the domains and to maintain their independent functions. Rigid linkers exhibit relatively stiff structures by adopting a-helical conformations or by containing multiple Pro residues. Examples of some rigid linkers are: (EAAAK)n (SEQ ID NO: 43) and (XP)n, with X designating any amino acid, preferably Ala, Lys, or Glu.

[0099] Additionally, or in the alternative, a linker for use in PM-Cld fusion proteins described herein can be an in vivo cleavable linker. Flexible and rigid linkers represent stable linkers that covalently join functional protein domains together to act as one molecule throughout the in vivo processes that the component protein(s) are Attorney Docket No. 29539-0849WO1 involved in. This stable linkage between functional domains provides many advantages such as a prolonged plasma half-life (e.g., albumin or Fc-fusions). However, it also has several potential drawbacks, including, steric hindrance between functional domains, decreased bioactivity, and altered biodistribution and metabolism of the protein moieties due to the interference between domains. Under such circumstances, cleavable linkers are used to release free functional domains in vivo. This type of linker may reduce steric hindrance, improve bioactivity, or achieve independent actions / metabolism of individual domains of recombinant fusion proteins after linker cleavage. The design of in vivo cleavable linkers in recombinant fusion proteins is quite challenging. Unlike the versatility of crosslinking agents available for chemical conjugation methods, linkers in recombinant fusion proteins must necessarily be oligopeptides. For example, an in vivo cleavable disulfide linker (LEAGCKNFFPJQSFTSCGSLE) (SEQ ID NO: 44; the arrow indicates where cleavage occurs), based on the reversible nature of the disulfide bond, was designed for recombinant fusion proteins by Chen et al. (Biotechniques 49:513-518, 2010), and offered the advantage of generating a precisely constructed, homogeneous product by recombinant methods.

[0100] Nucleic acid constructs

[0101] Also described herein are nucleic acid constructs (also referred to herein as nucleic acid molecules) that encode PM-Cld fusion proteins and / or Cid enzymes of the present disclosure. Such nucleic acid molecules can encode functional proteins. A functional Cid has chlorite decomposition activity, and a functional PM-Cld is targeted to plasma membrane of cells for generation of oxygen on the cell surface. The nucleic acid molecules can include a nucleotide sequence shown herein. The nucleic acid molecule can include sequences encoding the functional protein (i.e., “the coding region” or “open reading frame”), as well as 5’ untranslated sequences. Alternatively, the nucleic acid molecule can include only the coding region, e.g., without any flanking sequences that normally accompany the subject sequence.

[0102] A PM-Cld fusion protein and / or Cid enzyme can include a protein sequence that is at least about 80% or more homologous to the entire length of a sequence as shown herein. For example, the sequence can be at least about 80% identical (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, Attorney Docket No. 29539-0849WO1

[0103] 95%, 96%, 97%, 98%, 99%, or 100% identical over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200, or more amino acids) in length.

[0104] Methods of alignment of sequences for comparison are well-known in the art. For example, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CAB IOS 4: 11 17; the local homology algorithm of Smith and Waterman (1981) J. Mol. Biol. 147: 195-7; the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443 453; the search-for-similarity-method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444 2448; the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873 5877.

[0105] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. When comparing two sequences for identity, it is not necessary that the sequences be contiguous, but any gap would carry with it a penalty that would reduce the overall percent identity. For blastn (aligning nucleotide sequences), the default parameters are Gap opening penalty=5 and Gap extension penalty=2. For blastp (aligning protein sequences), the default parameters are Gap opening penal ty= 11 and Gap extension penalty=l . For BLASTP, the defaults are wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix [see Henikoff and Henikoff, (1992) Proc Natl Acad Sci USA 89(22): 10915-10919] alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strand Attorney Docket No. 29539-0849WO1

[0106] For use in the present compositions and methods, nucleic acid molecules encoding PM-Cld fusion protein and / or Cid enzyme can be used that has been codon optimized for expression in the cell, e.g., human codon optimized for expression in human cells. Nucleic acids encoding PM-Cld fusion protein and / or Cid enzyme can include mRNA or cDNA encoding the proteins, and the nucleic acids can be naked or in an expression vector, e.g., comprising a sequence such as a promoter that drives expression of the protein. The sequence can, for example, be in an expression construct.

[0107] Also provided herein are fusion proteins and nucleic acid molecules encoding the fusion proteins that can be cleaved to separate the PM-Cld components following their expression as a single polypeptide (e.g., with the components separated by a protease cleavage site, a ribosomal skip sequence, or a 2A self-cleaving peptide sequence).

[0108] The fusion proteins can include one or more ‘self-cleaving’ 2A peptides between the coding sequences. 2A peptides are 18-22 amino-acid-long viral peptides that mediate cleavage of polypeptides during translation in eukaryotic cells. 2A peptides include F2A (foot-and-mouth disease virus), E2A (equine rhinitis A virus), P2A (porcine teschovirus-1 2 A), and T2A (thosea asigna virus 2 A), and generally comprise the sequence GDVEXNPGP (SEQ ID NO:5) at the C-terminus. See, e.g., Liu et al., Sci Rep. (2017) 7: 2193. Table 4 provides exemplary 2A sequences.

[0109] TABLE 4. Exemplary 2A sequences Attorney Docket No. 29539-0849WO1

[0110] Alternatively, or in addition, the fusion proteins can include one or more protease-cleavable peptide linkers between the coding sequences. A number of protease-sensitive linkers are known in the art, e.g., comprising furin cleavage sites RX(R / K)R, RKRR (SEQ ID NO: 11) or RR; VSQTSKLTRAETVFPDVD (SEQ ID NO: 12); EDVVCCSMSY (SEQ ID NO: 13); RVLAEA(SEQ ID NO: 14); GGGGSSPLGLWAGGGGS (SEQ ID NO: 15); TRHRQPRGWEQL (SEQ ID NO: 16); MMP 1 / 9 cleavage sequence PLGLWA (SEQ ID NO: 17); TEV Protease sensitive linkers comprising ENLYFQ(G / S) (SEQ ID NO: 18); Factor Xa sensitive linkers comprising I(E / D)GR; or LSGRDNH (SEQ ID NO: 19) which is cleaved by cancer-associated proteases matriptase, legumain, and uPA. See, e.g., Chen et al., Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369.

[0111] Calculations of identity between sequences are performed as follows. 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). 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. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into Attorney Docket No. 29539-0849WO1 account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0112] Recombinant Expression Vectors

[0113] Also provided herein are vectors, preferably expression vectors, containing a nucleic acid molecule encoding PM-Cld fusion protein and / or Cid enzyme as described herein. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid or viral vector. The vector can be capable of autonomous replication, or it can integrate into a host DNA. Viral vectors include, e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses.

[0114] A vector can include PM-Cld and / or Cid nucleic acid sequences in a form suitable for expression of the nucleic acid in a host cell. Preferably the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. The term “regulatory sequence” includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and / or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the present disclosure can be introduced into host cells to thereby produce PM-Cld and / or Cid.

[0115] The recombinant expression vector can be designed for expression of the PM- Cld and / or Cid in any eukaryotic cells. For example, PM-Cld and / or Cid can be expressed in animal cells, e.g., mammalian cells, e.g., human or non-human primate cells, cells of rodent (e.g., rat, mouse, or hamster, e.g. CHO or COS cells), rabbit, cat, dog, cow, horse, goat, or other non-human mammals, or insect cells (e.g., using baculovirus expression vectors); or in fungus, e.g., in yeast cells. Thus, the expression vector can be, e.g., a yeast expression vector, a vector for expression in insect cells, e.g., a baculovirus expression vector, or a vector suitable for expression in mammalian cells. When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Attorney Docket No. 29539-0849WO1

[0116] Simian Virus 40. For expression of the Cid at the plasma membrane, the PM-Cld can likewise be expressed in animal cells as described above.

[0117] Genetically Engineered Cells and Organisms

[0118] The present methods and compositions can be used in any eukaryotic cells or non-human eukaryotic organisms. The eukaryotic cells or non-human eukaryotic organisms can be engineered to comprise a nucleic acid molecule encoding PM-Cld as described herein and express a PM-Cld fusion protein from the nucleic acid molecule. In some instances, the eukaryotic cells or non-human eukaryotic organisms do not express a chlorite transporter, or do not express an exogenous chlorite transporter. The eukaryotic cells or non-human eukaryotic organisms can also be engineered to comprise a nucleic acid molecule encoding Cid as described herein and express a Cid enzyme from the nucleic acid molecule.

[0119] Thus, provided herein are host cells that have been engineered to express a PM-Cld and / or Cid as described herein, optionally expressed from a recombinant expression vector or from sequences homologously recombined into the host cell's genome. The terms “host cell” and “recombinant host cell” are used interchangeably herein. Such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications can occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0120] The cells can be, for example, animal cells, e.g., mammalian cells, e.g., human or non-human primate cells, rodent (e.g., rat, mouse, or hamster, e.g., CHO or COS cells), rabbit, cat, dog, cow, horse, goat, or other non-human mammals, or insect cells (e.g., using baculovirus expression vectors); or fungus, e.g., yeast cells. The cells can also be immortalized cells that can be kept in culture. Other suitable host cells are known to those skilled in the art, see, e.g., Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA. The cells can also be human CAR-T cells, i.e., T cells that express chimeric antigen receptors (CARs) (Aghajanian et al., Nature Metabolism 4: 163-169 (2022); Gumber and Wang. EBioMedicine. 2022 Mar;77: 103941; Sterner and Sterner, Blood Cancer J. 2021 Apr 6;11(4):69. Preferably, the host cells do not express an endogenous chlorite Attorney Docket No. 29539-0849WO1 transporter. In some instances, the host cells are not Saccharomyces cerevisiae, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe, Trichoderma reesei, Neurospora crassa, Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipitis, Pichia pastoris, Sporotrichum thermophile, Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas mobilis, Clostridium saccharoperbutylacetonicum, Clostridium phytofermentans, Clostridium thermocellum, Clostridium beijerinckii, Clostridium acetobutylicum, Clostridium botulinum, Clostridium butyricum, Clostridium diolis, Clostridium ljungdahlii, Clostridium aerotolerans, Clostridium cellulolyticum, Clostridium tyrobutyricum, Clostridium pasteurianum, Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca, Thermoanaerobacterium saccharolyticum, Yarrowia lipolytica, or Bacillus subtilis.

[0121] Vector DNA can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.

[0122] Also provided are uni- and multicellular transgenic eukaryotic organisms comprising at least one cell that expresses PM-Cld and / or Cid. In some instances, every cell in the organism expresses PM-Cld and / or Cid. In some instances, the cells in the uni- and multicellular transgenic eukaryotic organisms do not express a chlorite transporter, or do not express an exogenous chlorite transporter. The organism can be an animal; for example, a mammal, e.g., a human or a non-human mammal, such as a mouse. The organism can be an arthropod, e.g., an insect such as a fruit fly, or a worm such as Caenorhabditis elegans. The organism can also be a plant or protist, e.g., algae. Further, the organism can be a fungus, e.g., yeast. Methods for generating transgnic organisms are known in the art.

[0123] Methods of Use

[0124] The present methods can include maintaining the cells and organisms described herein in an environment that includes chlorite, e.g., levels of chlorite about Attorney Docket No. 29539-0849WO1 the normal environment for the cells or organisms. For example, for eukaryotic cells, e.g., in culture, the methods can include culturing the cells in a media comprising added chlorite, e.g., 1 nM to 5 mM chlorite, preferably at least 5 nM, 10 nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 1 pM, 5 pM, 10 pM, 50 pM, 70 pM, 75 pM, 100 pM, 150 pM, 200 pM, 250 pM, 300 pM, 350 pM, 400 pM, 450 pM, or 500 pM chlorite, or up to 1 mM, 2.5 mM, or 5 mM chlorite. For transgenic non-human uni- or multi-cellular eukaryotic organism, the methods can include maintaining the organisms in an environment comprising chlorite, e.g., an aqueous environment comprising chlorite, or a gaseous environment comprising chlorite, e.g., sodium hydrogen chlorite (NaHClCL). The chlorite can be, e.g., sodium chlorite (NaQCL), chlorous acid (HCIO2), or a heavy metal chlorite (Ag+, Hg+, T1+, Pb2+, Cu2+ or NH+4).

[0125] The present methods (e.g., SNORCL and Surface SNORCL) can be used as a genetic tool in research settings to acutely evolve oxygen on demand in cultured cells or in model organisms. For example, SNORCL can target Cid to different subcellular compartments for localized oxygen production, and Surface SNORCL can target PM- Cld to the plasma membrane for generation of oxygen at the cell surface. Such studies can provide insight into the biology of anoxia, as well as the toxicity of hyperoxia (Ast & Mootha (2019), Nature Metabolism l(9):858-860). SNORCLs and Surface SNORCLs can serve as genetic tools for studies of “causal metabolism,” specifically to evaluate the causal role of oxygen in processes or diseases of interest.

[0126] In particular, Surface SNORCL of the present disclosure can be used for targeting PM-Cld to surface (e.g., plasma membrane) of cells, wherein the PM-Cld is exposed to the exterior of the cell. The PM-Cld can catalyze formation of oxygen from chlorite (CIO2 ), thus generating oxygen on surface of cells. Oxygen generated on surface of cells can be referred to herein as pericellular oxygen. Thus, PM-Cld can generate oxygen (e.g., pericellular oxygen) on surface of cells by catalyzing formation of oxygen from chlorite (e.g., non-toxic doses of chlorite). For example, PM-Cld can generate oxygen (e.g., pericellular oxygen) on surface of cells by catalyzing formation of oxygen from about 1 nM to about 5 mM of chlorite (e.g., about 2.5 nm, 5 nM, 10 nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 1 Attorney Docket No. 29539-0849WO1 pM, 5 pM, 10 pM, 25 pM, 50 pM, 70 pM, 75 pM, 100 pM, 150 pM, 200 pM, 250 pM, 300 pM, 350 pM, 400 pM, 450 pM, or 500 pM chlorite; or up to 1 mM, 2.5 mM, or 5 mM chlorite). Preferably, PM-Cld can generate oxygen (e.g., pericellular oxygen) on surface of cells by catalyzing formation of oxygen from about 1 nM to about 500 pM chlorite. Thus, PM-Cld can be used for generation of oxygen on surface of cells, such as mammalian cells (e.g., human cells, non-human primate cells, and non-primate mammalian cells, such as cells of rodent (e.g., rat, mouse, or hamster, e.g., CHO or COS cells), rabbit, cat, dog, cow, horse, goat, or other non- human mammals), insect cells, or other host cells described herein. In some instances, PM-Cld can be used to produce oxygen at cell surface at rates that outcompete (e.g., is greater than) utilization of oxygen by the respiratory chain. For example, oxygen generation on surface of cells by PM-Cld can be greater than utilization of oxygen by the respiratory chain by 1.5-fold or more (e.g., by 1.75-fold, 2.0-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3.0-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4.0- fold, 4.25-fold, 4.5-fold, 4.75-fold, 5.0-fold, 5.25-fold, 5.5-fold, 5.75-fold, 6.0-fold, 6.25-fold, 6.5-fold, 6.75-fold, 7.0-fold, 7.25-fold, 7.5-fold, 7.75-fold, 8.0-fold, 8.25- fold, 8.5-fold, 8.75-fold, 9.0-fold, 9.25-fold, 9.5-fold, 9.75-fold, or 10.0-fold or more).

[0127] PM-Cld of the present disclosure can also be used for enhancing and / or restoring toxicity (e.g., phototoxicity) of one or more genetic singlet oxygen O2 (O21) generators, which can be useful for targeted ablation of cells. Genetic O21generators (e.g., miniSOG, miniSOG2.0) can produce toxic O21in response to blue light illumination and can thus be used for targeted ablation of cells. However, oxygen limitation (e.g., hypoxic condition) can reduce the toxicity (e.g., phototoxicity) of genetic O21generators. Oxygen generation on surface of cells by PM-Cld can overcome the problem of oxygen limitation and can enhance the activity of genetic O21generators, e.g., by enhancing and / or restoring phototoxicity, even under hypoxic conditions. Thus, oxygen generation on surface of cells by PM-Cld can be used for enhancing and / or restoring toxicity (e.g., phototoxicity) of one or more genetic singlet oxygen O2 (O21) generators (e.g., even under hypoxic conditions), and a combination of PM-Cld and one or more genetic O21generators can be used for targeted cell ablation.

[0128] PM-Cld of the present disclosure can also be used for regulating endogenous signaling. For example, oxygen generation on surface of cells by PM-Cld can Attorney Docket No. 29539-0849WO1 influence endogenous signaling. In particular, oxygen generation on surface of cells by PM-Cld can be used for regulating the levels of Hypoxia-Inducible Factor 1 -alpha (HIFla, a central transcriptional regulator of the hypoxic response in cells) and expression of genes that are directly or indirectly targeted by HIFla. For example, oxygen generation on surface of cells by PM-Cld can lead to reduction of HIFla levels (e.g., depletion of HIFla protein); downregulation of genes such as DNA damage-inducible transcript 4 (DDIT4), adrenomedullin (ADA / ), basic helix-loop- helix family member e40 (BHLHE40), and Homeobox protein al3 (H0XA13),' and / or upregulation of genes such as thioredoxin-interacting protein (TXNIP).

[0129] The SNORCL technology can also have many medical and biotechnological applications. For example, it could be delivered as a gene therapy to target tissues and alleviate hypoxia-mediated diseases. Alternatively, SNORCL and Surface SNORCL can be useful in boosting the activity of cellular therapies such as CAR-T, where hypoxia in the tumor microenvironment contributes to T cell exhaustion (Schurich et al., 2019). Organisms genetically modified to express PM-Cld may even promote survival in extra-terrestrial, anoxic zones (e.g., surface of Mars) where chlorite is present.

[0130] EXAMPLES

[0131] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0132] Example 1. A genetically encoded system for oxygen generation inside living human cells

[0133] We began by testing the expression of several naturally occurring Cid variants as well as those engineered for greater thermostability or subcellular localization (Netzer 2018). To facilitate expression and purification from human HeLa cells, Cid genes were engineered through codon optimization, deletion of predicted periplasmic targeting sequences, and incorporation of epitope tags at the termini least likely to impact enzyme activity as suggested by published pentameric and dimeric CLD structures. We tested enzymes from both lineage 1 (FLAG-M / Cld, FLAG-Dt / Cld) and lineage 2 (MrCld-FLAG), including one targeted to mitochondria (mito-MrCld- FLAG). We also used computational methods (16) to design four point mutations Attorney Docket No. 29539-0849WO1 predicted to improve M / Cld thermostability (FLAG- Cld4xMUT). In these preliminary screens we saw the greatest expression from N-terminally FLAG-tagged M7Cld (FIG. 1C), which became the focus of our study. Cells expressing FLAG- M7Cld appeared healthy, comparable to cells expressing GFP, without any obvious impact on cell morphology or growth..

[0134] We next sought to determine whether FLAG-M / Cld expressed in human cells grown in ambient conditions at sea level was properly assembled with its heme b cofactor. We cultured cells expressing FLAG-M / Cld and performed affinity purification under non-denaturing conditions. The purified enzyme is monodispersed, as shown by gel filtration chromatography, running at an apparent molecular weight of 248 kDa (FIG. 2A). SDS-PAGE analysis results in a clean, Coomassie-stained band at the expected molecular weight of 29 kDa (FIG. 2B). Given that MAC Id, as well as other Lineage I Cids, are known to form pentamers (Kostan 2010), we speculate that in our mild detergent conditions, the enzyme is running as a dimer of pentamers. The oxidized and reduced spectra, obtained by addition of ferricyanide or dithionite, respectively, confirms that the enzyme expressed in human cells incorporates a heme b cofactor (FIG. 2C). We quantified the heme concentration from the absorbance at 557nm of the reduced M / Cld sample, and assuming a heme extinction coefficient of 34.7 rnNf'cm'1(Paul 1953), we estimate 98% incorporation of heme in the purified M7CLD.

[0135] We next characterized the activity of this protein in human cell extracts. We permeabilized HeLa cells with digitonin and then performed a dose response experiment with addition of sodium chlorite. Doses spanning 10 uM to 1 mM were used, as previous studies have shown that higher chlorite concentrations lead to inactivation of the enzyme (Hofbauer 2014). We monitored oxygen evolution using an optical probe in a well-stirred, air saturated cuvette. We observe very fast and strong oxygen evolution in response to added sodium chlorite (FIG. 2D), consistent with what has been reported for bacterial expressed and purified enzyme. For the bacterial expressed M7Cld, a broad range of Kmvalues have been reported, ranging from 58-69 uM for the purified enzyme (Kostan 2010; reviewed in Hofbauer 2014), to as high as 15.8 mM in the original characterization of M7Cld in E. coli extracts (Maixner 2008). In our human digitonin-permeabilized cell extract, based on initial rates of oxygen evolution, we estimate that the Kmfor chlorite is 560 uM and the Vmax Attorney Docket No. 29539-0849WO1 is 0.37 umoles / second / 100,000 cells (FIG. 2E). Our kinetic parameters for human HeLa cell extracts expresing / Cld are far superior to those reported in bacterial extracts, but our observed velocity is less than what has been reported for the the purified enzyme, likely because of active oxygen consumption by these extracts and presence of HEPES and chloride which have been shown to have a detrimental impact on Cid activity (Freire 2015; Streit 2008). Regardless, these studies demonstrate that M / Cld enzymes can be safely expressed in human cells grown in standard cell culture conditions, they oligomerize, fully incorporate the heme b co-factor, and function in a highly robust manner with rapid production of oxygen. These studies demonstrate that M / Cld enzymes can be safely expressed in human cells grown in standard cell culture conditions. They oligomerize, fully incorporate the heme b co-factor, and in permeabilized extracts, function properly with rapid production of oxygen from chlorite.

[0136] For M / Cld to be useful in intact cells, sodium chlorite would have to transit through the plasma membrane at doses tolerated for the specific application. However, as chlorite is negatively charged and polar, it is not expected a priori to rapidly diffuse into cells across the plasma membrane. Nonetheless, previous studies have shown that at very high doses, chlorite compromises fitness and growth of cells due to its oxidant properties (Ali 2016). Chlorite is an oxidant, and at high doses, can damage human erythrocytes (Ali 2016). In yeast, a 4 mM dose is required to achieve 50% growth inhibition (Kwolek-Mirek 2011). In order to both verify that Cid was active in HeLa cells under normal growth conditions as well as confirm that the cells expressing / Cld were healthy in the presence of chlorite, we performed a three-day toxicity study of HeLa cells bathed in chlorite-containing growth media. In HeLa cells, we observed a 50% decrease in viability when cells were treated with ~2mM of sodium chloride for 3 days (FIG. 3A). However, the toxicity was alleviated by the expression of / Cld (FIG. 3B). These data suggest that at a high dose chlorite can enter HeLa cells over a three-day period, and that it is toxic in a way that can be alleviated by expression of M / Cld.

[0137] Using this three day toxicity assay, we screened for transporters that might promote uptake of chlorite into human cells. To our knowledge, no study has ever investigated chlorite transport, though transport activity for the polyatomic anions nitrate, nitrite, and chlorate have been reported. We expressed both wild-type and Attorney Docket No. 29539-0849WO1 activity boosting point mutants of nitrate transporters from A. thaliana, A. nidulans, H. polymorpha, and human, without any obvious boost in chlorite toxicity (data not shown). We then turned to the human sodium iodide symporter (NIS), encoded by SLC5A5 (Eskandari 1997). The human NIS is expressed as a homodimer on the basolateral membrane of thyroid follicular cells with a C-in, N-out topology, where it electrogenically concentrates iodide with symport of 2 Na+ions. Electrophysiological studies of the NIS in Xenopus oocytes shows it has broad transport activity for many anions, including chlorate (CIOs') with a Kmof 277 uM (Eskandari 1997). When we expressed the human NIS in HeLa cells, we observed a five-fold increase in the three- day toxicity of added sodium chlorite (FIG. 3C) that could be attenuated by M / Cld co-expression (FIG. 3D). Without being bound by theory, the most parsimonious explanation our results is that NIS promotes chlorite uptake into the HeLa cells, and M / Cld catalyzes its conversion from chlorite to molecular oxygen and chloride.

[0138] We also sought to determine whether we could detect oxygen evolution in intact cells using SNORCLs (FIG. 4A). We grew Hela cells expressing either FLAG- M / Cld or GFP, with or without the co-expression of NIS (FIG. 4B), for measurements of oxygen consumption rate (OCR) using the Seahorse XFe96 Analyzer. We anticipated challenges in being able to detect oxygen evolution by SNORCLs given that any oxygen it generates could rapidly equilibrate with the atmosphere, and second, mitochondria could actively consume it. After initial experiments at 21% oxygen (FIGs. 6A-6B), where we did observe modest but reproducible oxygen generation in a Cld-dependent manner, we performed these experiments in a 1% ambient oxygen environment (to prevent back diffusion) while treating cells with piericidin and antimycin (to block mitochondrial respiration).

[0139] Under these conditions, oxygen generation, as evidenced by a decline in apparent OCR, was immediately obvious and striking in cells co-expressing both M / Cld and NIS, where we saw robust oxygen production with with a clear dose response beginning with 1 mM chlorite (FIGs. 4C-4D). In these experiments maximal rates of oxygen evolution occurred during the first ten minutes, but then continued for more than a total of 30 minutes. Cells remained viable throughout the course of these Seahorse experiments even one hour after addition of the highest doses of chlorite (FIG. 6C). In separate experiments, found no dimunition in viability four hours following a 30 minute exposure to high does chlorite (FIG. 7). Attorney Docket No. 29539-0849WO1

[0140] Examination of the oxygen partial pressures from the Seahorse traces (FIG. 4E) clearly shows a chlorite-dose dependent oxygen evolution in these intact cells in a way that is boosted with co-expression of NIS, which is clearly important given that Cid protein levels appear slightly lower in cells co-expressing NIS (FIG. 4B). Partial pressure of oxygen reported by the Seahorse instrument (Fig. 4E) clearly shows a chlorite dose-dependent oxygen evolution in these intact cells in a way that is boosted by co-expressing NIS (FIG. 4E). Collectively these studies provide definitive proof that SNORCLs permit on-demand oxygen generation within living human cells.

[0141] To further confirm that oxygen generation was taking place inside the cell, we performed an independent set of experiments in which we measured oxygen with both permeabilized and intact cells (FIGs. 8A-8D). In this set of experiments, we again expressed FLAG-Af / Cld, and this time co-expressed either NIS or mCherry, the latter serving as a viral transduction and antibiotic selection control. In both cell lines, we saw robust protein expression of FLAG-Af / Cld (FIG. 8A). When the plasma membrane is permeabilized, both cell lines exhibit comparable dose responses to injected sodium chlorite (FIG. 8B). In intact cells, although we were able to generate oxygen pulses in both cell lines at a high dose of 5 mM chlorite, we observed oxygen evolution even with 500 uM or 1 mM of chlorite in NIS but not mCherry expressing cells (FIGs. 8C-8D). These studies further confirm that co-expressing the NIS facilitates the transport of the chlorite into cells.

[0142] Finally, we sought to determine whether we could genetically target the SNORCL system to different subcellular compartments (FIG. 5A). We introduced an N-terminal mitochondrial targeting sequence to FLAG-Af / Cld (mito-FLAG-Af / C / t / ) and compared it to FLAG-Af / Cld. These constructs successfully targeted the enzyme to mitochondria and the cytosol, respectively, based on immunoblot analysis of respective cell fractions (FIG. 5B). To determine whether the mitochondrial targeted Af / Cld can function, we performed Seahorse analysis in intact cells, and found that the mito-FLAG-Af / C / t / is also capable of generating oxygen in response to added chlorite (FIG. 5C). Examination of the oxygen partial pressures from the Seahorse instrument (FIG. 5D) confirms net generation of oxygen by mito-FLAG-Af / Cld when NIS is coexpressed. These experiments support that chlorite entering into the cell is able to be taken up by mitochondria, presumably via mitochondrial anion transporters. Attorney Docket No. 29539-0849WO1

[0143] Collectively these studies provide proof that SNORCL can be targeted to mitochondria to permit on-demand oxygen generation with spatiotemporal resolution.

[0144] Materials and Methods Used in Example 1

[0145] Sequences

[0146] GFP was obtained from Addgene #19319, pLJMl-eGFP. mCherry was from Addgene #32383, pcDN A3.1-P eredox -mCherry. All other sequences were custom designed and synthesized for use in this study.

[0147] Generation of cell lines stably expressing transgenes

[0148] Cid enzymes and sodium / iodide symporters were stably expressed in HeLa cells using lentiviral transduction. Briefly, gene constructs were custom synthesized in pUC57-Kan (GenScript) with Nhel and EcoRI restriction sites at the 5’ and 3’ ends, respectively. Cid cDNA was subcloned into the pLYSl lentiviral expression vector (Addgene #50057), while SLC5A5 cDNA was subcloned into pLYS5 (Addgene #50054). Construct sequences were verified by Sanger sequencing (Azenta). Lenti virus was generated in 293 T cells (ATCC #CRL-3216). 106cells were seeded per dish in 6 cm culture dishes, in 5 ml media. The next day, the cells were transfected using X-tremeGENE HP transfection reagent (Roche #6366244001) with 1 ug of lentiviral construct, along with 900 ng psPAX2 (Addgene #12260) and 100 ng pCMV-VSV-G (Addgene #8454) lentiviral packaging and envelope plasmids. After forty-eight hours, lentivirus was collected and passed through a 0.45 um polyethersulfone syringe filter (Whatman #6780-2504). For lentiviral transduction, 2xl05HeLa cells (ATCC #CCL-2) The next day, cells were treated with 8 ug / ml polybrene (Sigma #H9268) and transduced with 400 ul lentivirus. After 48 hours, cells were passaged and selected with 2 ug / ml puromycin (Gibco #A1113803) or 100 ug / ml hygromycin B (Sigma #H3274), as appropriate. Once fully selected, cells were maintained in puromycin or hygromycin B for an additional passage prior to use for subsequent experiments. HeLa cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco #11995-065) supplemented with 10% fetal bovine serum (FBS, Sigma #2442), IX GlutaMax (Gibco #35050061), and penicillin / streptomycin (Gibco #15140122). Cells were maintained in a 37°C, 5% CO2 incubator. Attorney Docket No. 29539-0849WO1

[0149] Immunoblot analysis

[0150] For Western Blots from HeLa cell lysates, cells were first washed with ice cold PBS, then lysed with ice cold 1% Triton lysis buffer () supplemented with protease / phosphatase inhibitor (Cell Signaling #5872). Lysates were clarified by centrifugation at 21,000 x g for 10 min, at 4C. Supernatants were transferred to clean microcentrifuge tubes on ice. Protein content was quantified by Bradford assay (BioRad #5000205). Samples were normalized to 1 ug / ul in lysis buffer with IX SDS sample buffer (2% SDS, 5% P-mercaptoethanol, 5% glycerol, 47.4 mM Tris HC1, 16.6 uM Bromophenol Blue, pH 6.8). Samples were heated for 5 min at 95C on a heat block, and cooled at room temperature before loading on SDS-PAGE gels. Samples were run on Tris-Glycine gels at 120 volts for approximately 2 hours, then transferred to PVDF membranes (Bio-Rad #1704157) using a Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked in 5% milk / TBST for 1 hour at room temperature. Membranes were probed with anti GFP (Abeam #ab6556), anti-FLAG (Cell Signaling #2368), or anti- P-tubulin (Cell Signaling #2128) diluted 1: 1000 in 5% milk / TBST, incubated overnight at 4C. HRP-conjugated donkey anti-rabbit (Cell Signaling #7074) secondary antibody was used at 1 : 10,000 dilution in 5% milk / TBST for 1 hour at room temperature. Membranes were washed 6 x 5 minutes with IX TBST before and after secondary antibody incubation. Membranes were incubated with Western Lightning Plus ECL substrate (PerkinElmer #NEL104001EA) for 3 minutes. Luminescence was detected using Amersham Hyperfilm ECL film (GE Healthcare #28906838) developed on an X-Omat 2000A Processor (Kodak).

[0151] Purification and biochemical characterization of NdCld expressed in human cells HeLa cells were harvested, washed in PBS, and resuspended in buffer A containing 300mM NaCl, 50mM HEPES pH7.4, 2% glycerol, cOmplete EDTA-free protease inhibitor cocktail (Roche), PMSF, and Benzoase (Millipore Sigma). Cells were lysed with 10 strokes of a tight Dounce homogenizer followed by a total of 90 seconds of sonication on ice. The suspension was centrifuged at 25,000xg for 1 hour and the resulting lysate was incubated with anti-FLAG M2 affinity gel (Millipore Sigma) for 90 minutes. The slurry was loaded into a gravity flow column, the flow through collected, and the resin washed with 20 column volumes of buffer A (without the protease inhibitors and nuclease). The protein was eluted using multiple Attorney Docket No. 29539-0849WO1 incubations of the resin in buffer A containing lOOug / ml 3X FLAG peptide. The collected protein was concentrated via Amicon 10KD centrifugal filters (Millipore Sigma), filtered, and then loaded onto a Superdex 200 Increase 5 / 150 GL gel filtration column (Cytiva) equilibrated with lOOmM NaCl, 20mM HEPES pH 7.4, and 0.2% glycerol. Sizing of the protein through gel filtration was accomplished by comparison to a gel filtration standard (Bio-Rad) run under identical buffer, flow rate, and temperature conditions.

[0152] Assessment of heme content in purified protein

[0153] Heme incorporation was measured through the pyridine hemochromagen assay (Barr and Guo, 2015). Spectra were collected using a Nanodrop One C. Equal volumes of purified / CLD (9.4 uM) and a solution of 0.2 M NaOH, 40% (v / v) pyridine, and 500 uM potassium ferricyanide were mixed to generate the oxidized spectra. Sodium dithionite was then added to a final concentration of 2.5 mM in order to obtain the reduced spectra. The heme concentration was then determined from the absorbance at 557 nm of the reduced / CLD sample using the heme extinction coefficient 34.7 mM'1(Paul et al, 1953). The calculated heme concentration, 4.6 uM, corresponded to 98% incorporation of heme in the purified 7CLD.

[0154] Steady state kinetics ofNdCld in permeabilized human cells

[0155] HeLa cells were pelleted at 800xg for 3 min, washed with PBS, pelleted again, and then resuspended in assay buffer (125 mM KC1, 2 mM K2HPO4, 1 mM MgCh, 2 OmM HEPES pH 7.2, 5 mM glutamate, 5 mM malate, and 0.01% digitonin) at a concentration of 5xl06cells / ml. Oxygen production was measured using a FireSting optical oxygen meter connected to a sensor vial. One ml of cell solution (5xl06cells) was used for each measurement. Measurements were performed under ambient air conditions with stirring of the cell solution. The reaction was initiated by adding sodium chlorite solution (prepared in assay buffer) to predetermined concentrations. The initial rates were determined from the resulting oxygen traces using up to 20 seconds of the linear portion of the trace via the ICEKAT web server (Olp, 2020). The means of 3 replicate rates were plotted against the chlorite concentrations to estimate the KM. Attorney Docket No. 29539-0849WO1

[0156] Three-day toxicity of sodium chlorite in human cells

[0157] HeLa cells cells were trypsinized, counted, and prepared at 105cells / ml in normal growth media. 1 M sodium chlorite stock solution was prepared fresh at the time of the assay in UltraPure dH2O, and diluted to 2X working concentrations in cell growth media. Cells were seeded in 24-well plates, with triplicate wells for each condition. 500 ul of each 2X chlorite / media preparation was first added to the plate. 500 ul of cell suspension (5xl04cells) was then added to each well. The plate was gently mixed, and cells were grown for 3 days in a 37°C / 5% CO2 incubator. After 3 days, cells were washed briefly with 500 ul of PBS, trypsinized with 250 ul TrypLE Express, and resuspended with 750 ul of normal growth media to 1 ml total volume. In wells containing a majority of visibly dead, floating cells, cells were resuspended by vigorously pipetting up and down rather than by trypsinization. 200 ul of each cell suspension was then quantified using a Vi-Cell BLU Cell Viability Analyzer (Beckman Coulter).

[0158] Measurement of oxygen and oxygen consumption rates in intact cells

[0159] For OCR and O2 measurements in HeLa cells using the Agilent Seahorse XFe96 system, cells were seeded at 1.5xl04cells in 80 ul / well in 96-well Seahorse cell culture plates, in DMEM (Gibco #11995-065) supplemented with 10% FBS (Gibco #26140-079) and penicillin / streptomycin (Gibco #15140-122). After 16-20 hours, 175 ml of HEPES buffered Seahorse DMEM supplemented with 10 mM glucose, 1 mM sodium pyruvate, and 2 mM L-glutamine (Agilent) was added, and the plate was transferred to a 37°C non-CO2 incubator for one hour. The Seahorse cartridge was hydrated according to the manufacturer’s protocol. Piericidin A (Enzo Life Sciences) + Antimycin A (Sigma) and sodium chlorite (Sigma) were prepared in Seahorse DMEM and added to the wells by injections during the Seahorse run. Three or four baseline respiratory rate measurements were taken, followed by sequential injections of Piericidin A+ Antimycin A (three or four measurements) and sodium chlorite (12 measurements). To confirm uniform cell numbers across cell lines and no striking changes in cell numbers over the course of a Seahorse experiment (Fig. 6C), after the Seahorse run 2 mg / mL Hoechst 33342 (Invitrogen) was added to each well and incubated for 10 min, and the plate was imaged on a BioTek Cytation 5 Cell Imaging Multi-Mode Reader. The total number of nuclei (a proxy for the cell Attorney Docket No. 29539-0849WO1 number) in each well was determined. To perform Seahorse measurement at 1% ambient oxygen, a XFe96 system was set up in a Coy O2 Control In Vitro Glove Box. Hydrated Seahorse cartridge, Seahorse DMEM, and other reagents were incubated at 1% ambient oxygen in the glove box overnight prior to the Seahorse experiment. During the Seahorse run at 1% ambient oxygen, the “Hypoxia mode” was used according to Agilent’s protocol. Freshly prepared sodium sulfite solution was loaded into the cartridge to provide a “zero” oxygen reference.

[0160] For permeabilized Seahorse OCR measurements, HeLa cells were seeded at 1.5xl04cells / well in 80 ul / well growth media and grown overnight at 37°C. Seahorse cartridges were hydrated overnight at 37°C, according to the manufacturer’s protocol. After 16-20 hours, cells were washed once with MAS buffer (70 mM sucrose, 220 mM mannitol, 5 mM KH2PO4, 5 mM MgCh, 2 mM HEPES, 1 mM EGTA, 0.2% FA- free BSA). Cells were then permeabilized with MAS buffer supplemented with 2 nM XF Plasma Membrane Permeabilizer (Agilent 102504-100) and 1 uM each of Piericidin A+Antimycin A. Upon assay start, six baseline respiratory rate measurements were taken, followed by injection of chlorite and twelve respiratory rate measurements after chlorite injection. Permeabilized Seahorse experiments were also performed at 1% ambient oxygen, in a Coy O2 Control In Vitro Glove Box as described above.

[0161] Example 2. Robust generation of oxygen at the surface of human cells via plasma membrane-targeted SNORCL

[0162] To achieve “Surface SNORCL”, we used the pDisplay™ system, which targets proteins to the plasma membrane via an N-terminal IgK secretion signal and a C-terminal transmembrane domain from PDGFR that allows membrane tethering. Structural modeling predicted such a transmembrane anchor would not hinder Cid pentamer formation or activity (FIG. 9B-9D). We confirmed proper localization of PM-Cld, using an anti M / Cld antibody, by its co-localization with the pan-cadherin plasma membrane marker in HEK293T, HeLa, and A375 cells (FIG. 9A, FIGs. 12A- 12B) Control cells expressing cytosolic Cid (cyto-Cld) displayed diffuse intracellular expression pattern (FIG. 9A, FIGs. 12A-12B). Western blotting with the NdCld- specific antibody confirmed expression (FIG. 9E). Furthermore, we verified that Attorney Docket No. 29539-0849WO1

[0163] PM-Cld that surface displays did not result in cleavage or release of anchorless Cid in the extracellular milieu (FIG. 9E).

[0164] We sought to confirm the activity of Surface SNORCL in intact cells. In stirred suspensions of PM-Cld expressing 293T cells whose endogenous respiration is blocked with the antibiotic Antimycin A, NaCICh injection led to a rapid increase in dissolved O2 levels (FIG. 9F). Biochemical characterization of PM-Cld yielded kinetic parameters (Km= 674 ± 278 pM; Vmax = 278 ± 33 nmol O2 min1per 5* 106cells) comparable to previously reported data (Markhard et al., 2022) (FIG. 9G). Surface SNORCL was similarly robust in A375 cells expressing PM-Cld (FIGs. 9H- 91). While gently permeabilizing the plasma membrane (see methods) dramatically increased O2 production in cyto-Cld expressing cells (19.6-fold rate increase; 37.75- fold total O2), there was no such boost in PM-Cld cells, indicating that the enzyme had been fully targeted to the plasma membrane, across 293T, A375, and HeLa cell lines (FIGs. 9J-9L). As expected, with large boluses of NaClO?, we observed autoinactivation of the Cid enzyme, likely due to bleaching of the heme enzyme by hypochlorite, which has been previously reported (Schaffner et al., 2015). Satisfyingly, repeated NaC102 injections confirmed quantitative CIO2 conversion to O2 prior to complete inactivation (FIG. 13).

[0165] Having seen that PM-Cld is robust in multiple cell types in culture, we sought to determine if its activity remained intact in vivo using xenograft mouse tumor model. We engineered an oncocytic thyroid carcinoma cell line (NCLHCC) (Gopal et al., 2023) to express PM-Cld. We confirmed in cell culture that PM-Cld was properly expressed (FIG. 9M) and capable of robust O2 generation (FIG. 9N). We implanted these cells subcutaneously, as previously reported (Gopal et al., 2023), and saw that tumor growth was unchanged by expression of Surface SNORCL. After ~6 weeks, we measured O2 in the tumor using an optical O2 needle sensor and could observe a 100-fold increase in intratumoral O2 in response to NaC102 intra-tumor injection. This striking O2 evolution was dependent on the expression of PM-Cld, as we saw no such increase with NaC102 injection alone (FIG. 90). Hence, Surface SNORCL appears fully functional in vivo.

[0166] To be useful, rates of O2 generation by Surface SNORCL should be comparable to rates of its consumption. The bulk of O2 consumption in cells is due to mitochondrial respiration, which can be inhibited with Antimycin A and Piericidin A Attorney Docket No. 29539-0849WO1

[0167] (inhibitors of mitochondrial complex III and mitochondrial complex I respectively) or accelerated with an uncoupler such as Bam 15 (FIG. 9P). Intracellular O2 dynamics were monitored in intact cells cultured in hypoxia at 1% O2 using the phosphorescent probe MitoXpress Intra (Agilent) (FIG 9Q). Acute injection of Antimycin A and Piericidin rapidly increased intracellular O2 from -0.4% to -1%, matching ambient levels. Subsequent activation of PM-Cld with NaCICh further elevated intracellular O2, reaching a maximum of -3% O2 over the course of 40 minutes before gradually returning to ambient levels. In contrast, DMSO-treated control cells returned to baseline within 20 minutes, while Baml5-treated cells displayed only minimal O2 accumulation due to enhanced respiratory O2 consumption (FIG. 9Q). Collectively, these studies show that Surface SNORCL can produce O2 at rates that outcompete its utilization by the respiratory chain.

[0168] Next, we sought to use PM-Cld to enhance the activity of miniSOG2.0, a genetic singlet oxygen O2 (O21) generator that produces O21in response to blue light illumination. miniSOG was originally developed to promote contrast electronmicroscopy (Shu et al., 2011) and has since enjoyed widespread applications in optogenetics (Lin et al., 2013). Another particularly interesting application is in targeted cell ablation (Qi et al., 2012; Xu & Chisholm, 2016); however, in this context, O2 limitation has been hypothesized to be the major limiting factor even under 21% O2 conditions (Pogue & Hasan, 1997). We sought to overcome this limitation by pairing it with SNORCL. We began by expressing miniSOG2.0 (Makhijani et al., 2017) in the mitochondria and cytosol. We varied blue light doses prior to monitoring cell growth over three days (FIG. 10A). Although miniSOG2.0 expression was comparable in cytosol and mitochondria (FIG. 14), we observed differential sensitization to blue light, with mitochondrial miniSOG2.0 sensitizing cells particularly strongly (60s to full growth prevention vs 240s for cyto- miniSOG2.0). The toxicity from miniSOG2.0 is dramatically attenuated in 1% O2 hypoxia (FIG. 10B). Generation of O2 by PM-Cld restored phototoxicity, and again this effect was more pronounced in cells expressing mitochondrially localized miniSOG2.0 (FIG. 10C). These findings indicate that miniSOG2.0 activity can be boosted by localized O2 generation with Surface SNORCL.

[0169] Having shown that Surface SNORCL can produce O2 that gets into the cell, an important question is whether this localized O2 production can be sensed by cells and Attorney Docket No. 29539-0849WO1 influence endogenous signaling. HIFla is a central transcriptional regulator of the hypoxic response, stabilized under low O2 conditions and degraded upon reoxygenation (Maxwell et al., 1999). HEK293T cells expressing PM-Cld were cultured under hypoxia (1% O2) to induce HIFla accumulation (FIG. HA). We then used Surface SNORCL to generate a transient pulse of O2 in response to added NaCICh. Western blot analysis revealed a sharp reduction in HIFla levels within 15 minutes, with partial recovery at 30 minutes and full restoration after >2 hours (FIG. 11 A). Inhibition of respiratory chain complex I with Pieri ci din and complex III with Antimycin A - which raises steady state levels of oxygen by blocking its consumption - also led to HIFla degradation, as expected (Hagen et al., 2003), (FIG. 9Q), albeit at a slower rate than under direct O2 generation with PM-Cld. We sought to determine whether any canonical HIF1 target genes show a correspondingly “fast response.” Using qRT-PCR, we measured canonical HIF1 targets BNIP3L, CA9, DDIT4 (FIG. 11B, FIG. 15). Although all of these are well established HIF targets, it is notable that only DDIT4 exhibited a rapid decrease and recovery, matching that of HIFla protein levels.

[0170] Materials and Methods Used in Example 2

[0171] Reagents and Resources (table) Attorney Docket No. 29539-0849WO1 Attorney Docket No. 29539-0849WO1 Attorney Docket No. 29539-0849WO1

[0172] Cell lines and culture conditions

[0173] HEK293T, HeLa and A375 cells were obtained from ATCC. NCI-HCC cells were provided by Dr Venkata R. Vantaku and Dr Sareh Parangi. All experiments were performed within 20 passages upon receipt from ATCC. Cells were maintained in humidified, CCL-controlled 37°C tissue culture incubators with O2 control at 1- 21% O2 as indicated. Experiments requiring hypoxic harvest of cells were conducted in Coy O2 / CO2 and Temperature controlled gloveboxes with humidified culture boxes. Cells were routinely cultured in DMEM (Gibco) with 25 mM glucose, supplemented with 10% fetal bovine serum (FBS - Gibco), 4 mM Glutamine, 1 mM sodium pyruvate, 50 ug / mL uridine (Sigma- Aldrich) and 100 U / mL penicillin / streptomycin (Gibco). When cells were infected with lentivirus the growth medium was supplemented with Polybrene (Sigma-Aldrich) and when selection of successfully infected cells was necessary, cells were selected using Puromycin (Gibco), Hygromycin (Gibco) or G418 (Gibco).

[0174] Lentivirus production

[0175] Stable expression of the indicated proteins was achieved using lentiviral transduction. PM-Cld was constructed using the pDisplayTM (ThermoFisher) system. Cyto-Cld is identical to previously described Cid (Markhard et al., 2022). miniSOG2.0 was synthesized (Azenta Genewiz). To produce lentivirus 3 x 106HEK293T cells were seeded into a 10 cm cell culture dish in 10 mL of DMEM high glucose supplemented with 10%FBS and 100 U / mL of Penicillin / Streptomycin. The following day 500 pL of transfection mixture was added per plate containing 50 pL of Fugene 6 (Promega), 4 pg of pMD2.G (Addgene 12259), 6 pg of Pax2 (Addgene 12260) and 10 pg of target plasmid in 500uL filled with Opti-MEM medium (Gibco). After 15 min of incubation at room temperature the mixture was added to the HEK293T cell culture plate and incubated for 2 days upon which supernatant was captured and filtered through a 0.45 pm filter and stored at -80°C.

[0176] Immunofluorescence microscopy

[0177] Localization of PM-Cld and cyto-Cld was conducted using a custom-made antibody for M / Cld (Yenzyme) and a pan-cadherin antibody (Sigma- Aldrich). Fluorescence images were obtained with a Nikon ti2-E inverted microscope equipped Attorney Docket No. 29539-0849WO1 with a CSU-W1 spinning disc confocal and Andor Zyla 2.3 CMOS camera using a 60x water objective. Cells were plated in glass bottom 96-well plates coated with Poly-L-Lysine and following 24 hours of growth were fixed and permeabilized with Formaldehyde and triton-X-100. Fluorescently conjugated secondary antibodies were purchased from Thermo-Fisher.

[0178] Oxygen measurements using FireSting probe

[0179] Cid activity assays in live cells were performed using a FireSting O2 probe (PyroScience GmbH). Cells were detached with TrypLE (Gibco), counted using a Vicell XR / XR Blu (Beckman), and stored at 37°C. Batches of 1-25 x 106cells were pelleted and resuspended in 1-5 mL of assay medium (HEPES-buffered, phenol red- free high-glucose DMEM with glutamine, pyruvate, 100 U / mL penicillin / streptomycin, and 50 pg / mL uridine).

[0180] Oxygen measurements were conducted at room temperature under constant stirring in an OXVIAL4 sensor vial, read out using an ADVIAL4 adapter ring and SPFIB-BARE Optical Fiber (PyroScience GmbH). The O2 sensor was calibrated according to the manufacturer’s instructions using ambient air, with readings recorded via a FireSting-02 (2- or 4-channel) meter.

[0181] Cells were supplemented with Antimycin A (Sigma-Aldrich) to inhibit electron transport chain O2 consumption and equilibrated for 2 min before injecting freshly prepared sodium chlorite to the indicated final concentrations. In some experiments, Bam 15 or Antimycin A was added 2 min post-injection. In others, cells were pre-treated with XFPMP (Agilent) for gentle permeabilization; pre-incubation was required before O2 measurement to ensure full permeabilization.

[0182] Xenograft mouse studies of cancer cell line expressing Surface SNORCL

[0183] A mouse xenograft model was generated as described previously (Gopal et al., 2023). All animal work was performed at Massachusetts General Hospital (MGH) in accordance with federal, local, and institutional guidelines. Mouse weights were tracked with digital scales, and tumor sizes were measured using digital calipers every week, with tumor volumes (TV) calculated from the formula volume = Yi (length x width2). Once tumors reached an approximate volume of 100 mm3, mice were randomized in equal numbers for control and NaClO? treatment groups. Mice were Attorney Docket No. 29539-0849WO1 prepared for pCh measurements by injecting ketamine / xylazine at the anesthesia regime. For in vivo optical recording of pCh, an aseptic calibrated optic fiber OXIMP250 (Pyro Science) was carefully inserted into the subcutaneous tumors to record the pCh concentration in the tumor microenvironment until stabilization. Once the pO? values were stabilized, we performed the intratumoral injection of 25mg / kg bodyweight NaCICh in 50 uL sterile saline solution or control saline solution. pO? levels were measured for the next 60 minutes without interruption.

[0184] Poly-acrylamide gel electrophoresis and protein immunoblotting

[0185] Preparation of cell extracts for polyacrylamide gel electrophoresis and protein immunoblotting was largely conducted as previously described (Ast et al., 2019). For preparation of hypoxic samples, lysis buffer was supplemented with 10 pM MG-132 (ThermoFisher).

[0186] MitoXpress Intra probe

[0187] To measure intracellular O2 levels, HEK293T cells were seeded in black, clear bottom TC-treated 96-well plates (Coming) in regular high glucose DMEM with supplements (Gibco). After overnight incubation, cells were loaded with MitoXpress Intra (Agilent) according to the manufacturer’s instructions. The MitoXpress Intra probe signal was read out using a Cytation 5 (Agilent / BioTek) cell imaging multimode reader with a phosphorescence lifetime detection module used according to the manufacturer’s instructions. Calibration of the phosphorescent probe and measurements were conducted inside an oxygen controlled Coy glovebox (Coy) according to the manufacturer’s instructions. qRT-PCR

[0188] Approximately 5 x 106cells were collected per sample, on ice and washed with ice cold PBS (Gibco). Pellets were snap-frozen in liquid nitrogen and stored at - 80°C. RNA was extracted using the RNeasy kit (Qiagen). Reverse transcription was conducted with random primers (ThermoFisher) and MLV-RT (Promega) supplemented with RNaseOUT (ThermoFisher). qPCR was performed using TaqMan technology probes (ThermoFisher) as indicated and read-out using a CFXOpus384 thermocycler (BioRad). Attorney Docket No. 29539-0849WO1

[0189] Incucyte growth assays

[0190] To determine light sensitivity of miniSOG2.0 expressing cells, cells were plated in black, clear bottom TC-treated 96-well plates (Coming) and incubated overnight under the indicated O2 conditions. When indicated cells were exposed to 100 pM NaCICh prior to illumination using the Cytation 5 (Agilent / BioTek) equipped with a GFP-filter cube (1225101 Agilent) which allows for excitation at 469 / 35 nm. Cells were then transferred to an cell culture incubator containing an Incucyte SX5 (Sartorius) brightfield images were captured every 1.5-3 hours for the duration of the experiment. Masks were created to accurately determine growth area. When indicated that illumination occurred under hypoxic conditions cells otherwise cultured at 21%O2, or room air, were transferred to a humidified, CO2 -controlled glovebox kept at 1%O2 and equilibrated for approximately 1.5 hours prior to illumination, executed by a Cytation 5 imaging system kept within a 1% O2 atmosphere in a glove box (Coy). After illumination cells were transferred back to 21% O2 and monitored via incucyte as described above.

[0191] Exemplary Sequences

[0192] Provided herein are exemplary human codon optimized DNA and corresponding protein sequences used in this study, which provide examples of sequences usable in the methods and compositions described herein (optionally omitting the FLAG (DYKDDDDK (SEQ ID NO: 1)) sequence and any linkers, e.g., GS-rich linkers (GGSGGSGGS (SEQ ID NO:2))). All other sequences were custom designed and synthesized for use in this study.

[0193] SEQUENCE TABLE Attorney Docket No. 29539-0849WO1 Attorney Docket No. 29539-0849WO1 Attorney Docket No. 29539-0849WO1 Attorney Docket No. 29539-0849WO1 Attorney Docket No. 29539-0849WO1 Attorney Docket No. 29539-0849WO1 Attorney Docket No. 29539-0849WO1 Attorney Docket No. 29539-0849WO1 Attorney Docket No. 29539-0849WO1

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[0264] OTHER EMBODIMENTS

[0265] 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 Attorney Docket No. 29539-0849WO1 following claims. All references, scientific articles, patent publications, and any other documents cited herein are hereby incorporated by reference for the substance of their disclosure.

Claims

Attorney Docket No. 29539-0849WO1WHAT IS CLAIMED IS:

1. A fusion protein comprising: a bacterial or archaeal chlorite:O2 lyase (Cid), a secretory signal peptide, and a cell membrane tethering domain, wherein the secretory signal peptide directs the Cid to plasma membrane of a cell and the cell membrane tethering domain tethers the Cid enzyme to the plasma membrane.

2. The fusion protein of claim 1, wherein the Cid is from Nitrospira defluvii ( VCld), Dechloromonas aromatica (Z zCld), or Nitrobacter winogradskyi (MrCld).

3. The fusion protein of claim 1 or 2, wherein the Cid lacks a functional periplasmic targeting sequence.

4. The fusion protein of any one of claims 1-3, wherein the Cid comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 3.

5. The fusion protein of any one of claims 1-4, wherein the Cid is encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 36.

6. The fusion protein of any one of claims 1-5, wherein the secretory signal peptide is positioned at the N-terminus of the Cid.

7. The fusion protein of any one of claims 1-6, wherein the secretory signal peptide is encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 34.

8. The fusion protein of any one of claims 1-6, wherein the secretory signal peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of any of SEQ ID NOs: 45-58.Attorney Docket No. 29539-0849WO19. The fusion protein of any one of claims 1-8, wherein the cell membrane tethering domain is positioned at the C-terminus of the Cid.

10. The fusion protein of any one of claims 1-9, wherein the cell membrane tethering domain comprise a transmembrane domain.

11. The fusion protein of any one of claims 1-10, wherein the cell membrane tethering domain is encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 38; or wherein the cell membrane tethering domain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of any of SEQ ID NOs: 59-61.

12. The fusion protein of any one of claims 1-11, wherein the Cid is directly fused to the secretory signal peptide and / or the cell membrane tethering domain.

13. The fusion protein of any one of claims 1-11, further comprising one or more purification tags and / or linkers.

14. The fusion protein of claim 13, wherein the one or more purification tags and / or linkers are positioned between the secretory signal peptide and the Cid.

15. The fusion protein of claim 13 or 14, wherein the one or more purification tags and / or linkers are positioned between the Cid and the cell membrane tethering domain.

16. The fusion protein of any one of claims 13-15, wherein the fusion protein comprises one or more of HA tag, FLAG-tag, and 3xGGS linkers.

17. The fusion protein of claim 16, wherein the one or more of HA tag, FLAG- tag, and 3xGGS linkers are positioned between the secretory signal peptide and the Cid.Attorney Docket No. 29539-0849WO118. The fusion protein of claim 16 or 17, wherein the one or more of HA tag, FLAG-tag, and 3xGGS linkers are encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 35.

19. The fusion protein of any one of claims 13-18, wherein the fusion protein comprises one or more Myc tag.

20. The fusion protein of claim 19, wherein the one or more Myc tag is positioned between the Cid sequence and the cell membrane tethering domain.

21. The fusion protein of claim 19 or 20, wherein the one or more Myc tag is encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 37.

22. The fusion protein of any one of claims 1-21, wherein the fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 40, optionally omitting one or more purification tags and / or linker sequences.

23. A nucleic acid molecule encoding the fusion protein of any one of claims 1-22.

24. The nucleic acid molecule of claim 23, wherein the nucleic acid molecule comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 39, optionally omitting nucleotide sequence encoding one or more purification tags and / or linker sequences.

25. The nucleic acid molecule of claim 23 or 24, wherein the nucleotide sequence is codon optimized for expression in a eukaryotic cell, optionally in an animal cell, e.g., a human cell.Attorney Docket No. 29539-0849WO126. A vector comprising the nucleic acid molecule of any one of claims 23-25.

27. A host cell comprising the vector of claim 26, and optionally expressing the fusion protein of any one of claims 1-22.

28. The host cell of claim 27, wherein the host cell is an animal cell, optionally a mammalian cell, e.g., a human cell.

29. An isolated eukaryotic cell expressing the fusion protein of any one of claims 1-22.

30. The isolated eukaryotic cell of claim 29, wherein the Cid is expressed on the cell surface.

31. The isolated eukaryotic cell of claim 29 or 30, wherein the isolated eukaryotic cell is an animal cell.

32. The isolated eukaryotic cell of claim 31, wherein the animal cell is a mammalian cell.

33. The isolated eukaryotic cell of claim 32, wherein the mammalian cell is a human cell, optionally a CAR-T cell.

34. A method for generating oxygen on the surface of a eukaryotic cell, the method comprising culturing the isolated eukaryotic cell of any one of claims 29-33 in a medium comprising chlorite, optionally wherein the medium comprises about 1 nM to about 500 pM chlorite.

35. The method of claim 34, wherein the rate of oxygen generation is greater than the rate of oxygen utilization by respiratory chain of the cell.Attorney Docket No. 29539-0849WO136. A method for enhancing and / or restoring toxicity of a genetic singlet oxygen (O21) generator in a cell, the method comprising expressing the genetic O21generator with the vector of claim 26 in the cell.

37. A transgenic non-human uni- or multi-cellular eukaryotic organism comprising the isolated eukaryotic cell of any one of claims 29-33.

38. The transgenic non-human uni- or multi-cellular eukaryotic organism of claim 37, which is a worm, a plant, or a mouse.

39. A method of generating oxygen in a transgenic non-human uni- or multicellular eukaryotic organism, the method comprising maintaining the organism of claim 37 or 38 in an environment comprising chlorite, optionally wherein the environment comprises about 1 nM to about 500 pM chlorite.

40. A method of generating oxygen on the surface of a eukaryotic cell, the method comprising culturing the host cell of claim 27 or 28 in a medium comprising chlorite, optionally wherein the medium comprises about 1 nM to about 500 pM chlorite.

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