Methods and compositions for editing phd

Abstract

The present invention relates to systems and compositions for editing of prolyl hydroxylase domain-containing protein (PHD) gene, and method of inhibiting PHD (e.g., for use in treating retinal degenerative diseases). Particularly the present disclosure provides systems, compositions, and methods for editing and / or ablation of the prolyl hydroxylase domain-containing protein 2 (PHD2) gene.

Description

METHODS AND COMPOSITIONS FOR EDITING PHDFIELD

[0001] The present invention relates to systems and compositions for editing a prolyl hydroxylase domain-containing protein (PHD) gene, e.g., PHD2, and methods of inhibiting PHD (e.g., for use in treating retinal degenerative diseases).CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 553,964, filed February 15, 2024, the content of which is herein incorporated by reference in its entirety.SEQUENCE LISTING STATEMENT

[0003] The content of the electronic sequence listing titled COLUM_41938_601_SequenceListing.xml (Size: 84,371 bytes; and Date of Creation: February 13, 2025) is herein incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support under EY033770 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0005] Retinitis pigmentosa (RP) is a group of inherited retinal dystrophies that affects up to 1 in 4000 people worldwide. Retinitis pigmentosa causes an initial phase of rod photoreceptor loss, followed by secondary loss of cone photoreceptor cells. Individuals with RP experience symptoms such as night blindness, tunnel vision, and total blindness. Together, these vision changes severely impact patients by limiting their ability to conduct activities required for daily living independently. Patients with degenerating photoreceptors, including those with RP, currently have limited therapeutic options. Although defects in over 80 genes are linked to RP, currently only patients with a recessive mutation in RPE65 can opt for precision gene supplementation therapy. Alternative therapeutic strategies that potentially can influence multiple types of photoreceptor degenerative disorders, including RP and age-related macular degeneration (AMD), are necessary.SUMMARY

[0006] Provided herein are systems and methods for increasing photoreceptor survival and resistance to stress. Such therapeutic intervention has the potential to be effective against retinal disorders caused by a diverse array of strain or genetic deficiencies.

[0007] In one aspect provided herein are methods for increasing retinal cell survival and / or preserving retinal function.

[0008] In some embodiments, the methods comprise contacting a retinal cell with a prolyl hydroxylase domain-containing protein (PHD) gene editing system, or one or more nucleic acids encoding thereof, wherein the PHD gene editing system ablates or truncates the PHD gene. In some embodiments, the methods comprise contacting a retinal cell with a prolyl hydroxylase domain-containing protein 2 (PHD2) gene editing system, or one or more nucleic acids encoding thereof, wherein the PHD2 gene editing system ablates or truncates the PHD2 gene.

[0009] In some embodiments, the PHD gene editing system or the PHD2 gene editing system comprises a CRISPR / Cas gene editing system. In some embodiments, the CRISPR / Cas gene editing system comprises one or more guide RNAs (gRNAs) targeting PHD2. In some embodiments, the one or more gRNAs are each configured to target exon 1 of the PHD2 gene. In some embodiments, the CRISPR / Cas gene editing system comprises two gRNAs, each configured to target different sequences in exon 1 of the PHD2 gene. In some embodiments, the CRISPR / Cas gene editing system comprises two gRNA of SEQ ID NOs: 11 and 12. In some embodiments, the one or more gRNAs are provided in a crRNA array.

[0010] In some embodiments, the methods comprise contacting the cell with an inhibitor of prolyl hydroxylase domain-containing protein (PHD). In some embodiments, the inhibitor of PHD targets any one or all of PHD 1, 2, and 3 isozymes.

[0011] In some embodiments, the inhibitor of PHD decreases the expression or level of PHD or decreases the activity of PHD.

[0012] In some embodiments, the inhibitor is a gene editing system which specifically targets and ablates a gene expressing PHD. In some embodiments, the gene editing system is a CRISPR- Cas9 system. In some embodiments, the gene editing system targets exon 1 of PHD2.

[0013] In some embodiments, the retinal cell is a rod cell and / or a cone cell.

[0014] In some embodiments, the cell is in a subject. In some embodiments, the method comprises administering the PHD gene editing system or PHD2 gene editing system, or one ormore nucleic acids encoding thereof, to the subject. In some embodiments, the method comprises administering the inhibitor of PHD, or one or more nucleic acids encoding thereof, to the subject. In some embodiments, the administration is by intravitreal or subretinal injection.

[0015] In some embodiments, the subject has or is suspected of having one or more retinal degenerative diseases. In some embodiments, the one or more retinal degenerative diseases comprise retinitis pigmentosa (RP), age-related macular degeneration (AMD), glaucoma, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Lewy body dementia, or combinations thereof.

[0016] In some embodiments, the one or more nucleic acids comprise a recombinant adeno- associated viral (AAV) vector. In some embodiments, the AAV vector is an AAV8 vector.

[0017] Also provided herein are systems and compositions comprising a gene editing system, or one or more nucleic acids encoding thereof, which specifically targets and ablates a gene expressing PHD. In some embodiments, the gene editing system is a CRISPR-Cas9 system (e.g., a Cas9 nuclease and one or more guide RNA). In some embodiments, the gene editing system targets exon 1 of PHD2.

[0018] Further provided herein are gene editing systems, or one or more nucleic acids encoding thereof, for ablating a prolyl hydroxylase domain-containing protein 2 (PHD2) gene, comprising: an RNA guided nuclease; and at least one gRNA configured to target exon 1 of the PHD2 gene.

[0019] In some embodiments, the one or more nucleic acids comprise a recombinant adeno- associated viral (AAV) vector. In some embodiments, the AAV vector is an AAV8 vector.

[0020] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and related figures.BRIEF DESCRIPTION OF THE FIGURES

[0021] FIGS. 1A-1E show that a PHD inhibitor enhances photoreceptor survival and preserves retinal function by increasing retina glycolysis. Pde / / ’'1620- '1620~ mice were fed the PHD inhibitor, FG-4592, at one dose every two days from P5. (FIG. 1A) Metabolites were extracted from the retinae of mice at P23.13C-labeled glycolytic intermediates increased in retinae from mice in the treatment group compared to those in the control group. Minimal effects were detected in TCA cycle intermediates and mitochondrial oxidation metabolites, shown here as treat vs. control. *P<=0.05, **P<=0.01. N=5-6 per group. (FIG. IB) H&E was performed toanalyze retinae from the Pdeb'’11620 1620~ mouse model at P35, with a representative section comparing the treatment group and control group at 500 pm from the optic nerve head. The green bar indicates the outer nuclear layer (ONL). Scale bar = 25 pm. (FIG. 1C) Spider plot analysis of ONL thickness. Bars indicate S.E.M. *P<=0.05, **P<=0.01, ***P<=0.001 n=6 per group. (FIG. ID) Pci e / / ’’1620~H62t'~ mice were subjected to three types of serial ERG recordings: scotopic (rod-specific), maximal (rod and cone), and photopic (cone-specific) at P28. Single tracings of the 3 -step ERG were shown. Blue represents the ERG tracing from a mouse with FG4592, while the gray tracing is from sham-fed control. (FIG. IE) Analysis of the amplitude of b-wave as shown in FIG. ID. Each dot or diamond represents the average ERG amplitude of both eyes per mouse. Bars indicate S.E.M. *P<=0.05, n=6 per group.

[0022] FIGS. 2A-2C show PHD deficiency up-regulates key regulators of glycolytic metabolism in photoreceptors. Experimental (PHD2;Pcle6p,l620<~,l620~;Pde / y’C'erRT2 / +) and control (PHDr, r,;Pde6[) H620Q / H620Q ,p^e^CreERT2 / +^mjce weretreated with tamoxifen or sham solution, respectively, for three consecutive days (P8, P9, and PIO). Mice were sacrificed at postnatal three weeks, and retinae were collected and snap-frozen in liquid nitrogen until further processing. (FIG. 2A) mRNA expression of Hifla and Hif2a and the downstream glycolytic targets (Glutl, 2, 3, 5, Hkl, 2, Pfkp, Pfkm, Pfkl, Aldoa, Pgkl, Enol, Pkml, 2, Ldha, b and Pdkl) were quantified to assess changes before and after PHD was ablated. p-Actin was used as the internal control. N = 4-5 per group. *P<=0.05, **P<=0.01, ***P<=0.001, ****P<=0.0001. (FIG. 2B) Representative immunoblots of glycolytic metabolism enzymes and regulators in the retinae of treated and untreated mice at P21 (before the onset of degeneration) to detect HIF1A, HIF2A, GLUT1, HK2, and LDHA protein levels. p-Actin was used as a loading control. Membrane was stripped and re-probed for all targets. (FIG. 2C) Quantitative analysis of protein levels shown in FIG. 2B. Bars indicate S.E.M. *P<=0.05, **P<=0.01, n=3 per group.

[0023] FIG. 3 shows loss of PHD enhances glycolysis and diminishes mitochondrial oxidation in PDE6-deficient retinae. Eyes from control PHD11'1and PHD ftliomozygous for Pde6l>'1620- '1620~ ) mice were dissected at P21 before retinal degeneration. Retinae were isolated under ambient illumination, incubated with 5 mM U-13C glucose, and harvested at 30s and 90s time points. Metabolites were extracted with 80% methanol, derivatized and quantified by GC- MS. Flux of carbons from U-13C glucose through glycolysis is faster in the PF / D-deficient retinae and TCA cycle activity is slower than controls. The bar graphs in the upper right sectionof the figure show that the average rates over 40 minutes at which glucose is consumed and at which lactate is exported are not significantly different between experimental and control retinae. Axis units are indicated in the center field, with the Y-axis representing picomoles or pg of protein with the X-axis representing time (seconds). Bars indicate S.E.M. *P <= 0.05, **P<=0.01, n=6-9 per group.

[0024] FIGS. 4A-4H show upregulation of glycolysis through ablation of PHD enhances cone survival in a preclinical Pde6 / 3H620Q / }i62W^ RP model. PHD deficiency improves rod and cone survival in the mutant background. Experimental (PHD2; Pde6 / 3H620Q / H620Q;Pde6' / reERT2) and control (PHDri ri; Pde6[}H620Q / H620Q; Pde6yCreERT2) mice were treated with tamoxifen or sham solution for three consecutive days (P9, P10, and Pl 1). (FIGS. 4A-4C) Analysis of amplitudes of electroretinogram tracings comparing experimental and control mice. Mice were subjected to three types of serial ERG recordings: scotopic rod (FIG. 4A), maximal rod and cone (FIG. 4B), and photopic cone (FIG. 4C) at 6, 8, and 10 weeks postnatally. Experimental mice with precise PHD 1,2, 3 ablation in rod photoreceptors were shown in red, while the signal from control mice is in gray. The average ERG amplitude of both eyes per mouse was used for analysis. Bars indicate S.E.M. *P<=0.05, **P<=0.01, ***P<=0.001, ****p<=0.0001, n=5-6 in each group per time point. (FIG. 4D) Representative H&E-stained central retinal sections from experimental and control mice at 4 and 6 weeks at a distance of 500 pm from the optic nerve head. Yellow bars indicate ONL thickness. Scale bar = 25 pm. (FIG. 4E) Spider plot analysis of ONL thickness at 4 weeks. *P<=0.05, **P<=0.01, ***P<=0.001, n=6 per group. (FIG. 4F) Spider plot analysis of ONL thickness at 6 weeks. *P<=0.05, **P<=0.01, ***P<=0.001, n=6 per group. (FIG. 4G) Peanut agglutinin (PNA) staining of cones in 12-week- old PHD11'1control and PHD " (deficient) mice. The inset shows representative central retinal cones. (FIG. 4H) Bar chart of cone cell counts, quantified by percent area of the total area measured in both groups in FIG. 4G. Bars indicate S.E.M. **P<=0.01, n=3 in each group.

[0025] FIGS. 5A-5F show ablation of PHD2 with gRNAs is sufficient for Hifla augmentation and target engagement in vitro. Schematic summary of the outcomes produced by gRNA therapeutic deletion. (FIG. 5A) The gRNAs used target exon 1 in human PHD2 sequence. (FIGS. 5B-5C) 95% of nonhomologous end joining (NHEJ) insertions and deletions (indels) resulted in a frameshift mutation in both mouse N2A (FIG. 5B) and human HEK293 (FIG. 5C) cells. Indels that are indivisible by 3 represent a successful insertion of a frameshift-mediated stop codon,truncating gene function. Unsuccessful edits are indicated with an Asterix (*) and account for around 5% of the cases in both N2A (FIG. 5B) and HEK293 (FIG. 5C) cells. (FIG. 5D) Immunoblotting was performed and the level of PHD2 from mouse N2A cells with control (PX459) or plasmids containing gRNAs targeting PHD2 was determined. p-Actin was used as a loading control. (FIG. 5E) Immunoblot was performed and the level of PHD2 from human HEK293 cells with control (PX459) or plasmids containing gRNAs targeting PHD2 was quantified. p-Actin was used as a loading control. After transfection with scramble gRNAs or gRNAs targeting PHD2, HEK293 cells were cultured in standard media. Immunoblots revealed lower levels of PHD2 after gRNA therapeutic gene deletion of PHD2 compared to scramble gRNA controls. (FIG. 5F) Increased expression of LDPIA, PDK1, GLUT1, and GLUT3 (glycolytic markers of the Warburg effect) after ablation of PHD2 in normoxic conditions. Control transduced with plasmids containing scramble gRNAs. Bars indicate S.E.M. **P<=0.01, ***P<=0.001, n = 3 in each group.

[0026] FIGS. 6A-6C show that approximately 35% of CD73+rod photoreceptors were transduced by AAV8::hGRKl-GFP. Mouse retinae receiving a subretinal injection (FIGS. 6A- 6C) of AAV8::hGRKl-GFP for 1 month were dissociated, labeled with the rod photoreceptor marker anti-CD73, and analyzed by flow cytometry. BL6 wild type mice were used here as a control. (FIG. 6A) Representative results in control mice with no injection and (FIGS. 6B-6C) in two injected mice. The GFP+ / CD73+cells are gated with the visible blue square and labeled as "P3"; the GFP7CD73+cells were gated with the red square and labeled as "P4". X-axis: CD73; Y-axis: GFP.

[0027] FIGS. 7A-7H show AAV8::U6-gRNAs_ / :, / 7D2 CRISPR-Cas9 therapy improves photoreceptor function and structure in Pde6 / 3H620Q / Cas9 arRP (FIGS. 7A-7C) and Rhocll0R>+adRP (FIGS. 7D-7H) mouse models. (FIG. 7A) Injection with a single dual-vector, AAV8::U6- gRNAs_PHD2;hGRKl-GFP, allowed marking of ventral subretinal transduction sites. Cas9 expression was transgenic in this line. (FIG. 7B) Representative global electroretinography (ERG) traces of AAV-treated right eyes (red) and untreated left fellow eyes (black) from the experimental groups. There were improvements in the scotopic ERG b-wave, mixed rod-cone ERG a- and b-waves, and photopic ERG b-wave recordings (pV) from the AAV2 / 8(Y733F)- gRNA transduced eyes compared with uninjected fellow eyes at 8 weeks of age. The subretinal injection was performed on one eye of each mouse at Pl 4. ERG was performed 8 weeks post-injection. (FIG. 7C) Gray bars represent untreated fellow eyes, red bars represent vector- transduced eyes, and black dots represent each mouse after virus treatment. AAV8-transduced and uninjected fellow eyes were compared at each time point. *P < 0.05, **P < 0.01; n = 4 for all groups. (FIG. 7D) Co-injection of two vectors AAV8::U6- gRNAs_PHD2;hGRKl-GFP and AAV8::hGRKl-Cas9 allowed marking of subretinal transduction sites in dominant Rhocn0R / +at 1 -month post-injection. (FIG. 7E) Representative global ERG traces of AAV-treated right eyes (red) and untreated left, fellow eyes (black) from the experimental groups. The subretinal injection was performed on one eye of each mouse at P28. There were statistically significant improvements in the scotopic ERG b-wave, mixed rod-cone ERG a and b waves, and photopic ERG b-wave recordings (pV) from the AAV2 / 8(Y733F)-gRNA transduced eyes compared with the uninjected fellow eyes at 5 months of age. Gray bars represent untreated fellow eyes, whereas red bars represent vector-injected eyes (black dots represent each mouse after virus treatment). (FIG. 7F) AAV8-transduced and uninjected fellow eyes were compared at each time point. **P<=Q.Q1, ***P <=0.001; n = 4 for all groups. (FIG. 7G) H&E-stained retinal sections taken from peripheral Rhocn0R / +retina transduced ventrally with AAV8::U6-gRNAs_F, / / D2 in the right eye. Yellow bar = ONL thickness. Scale bar (black, top left) = 25 pm. (FIG. 7H) Quantification of ONL thickness in Rhocn0R / +mice injected with the dual AAV8 compared to the non-injected fellow eye as seen in FIG. 7G. Bars indicate S.E.M. *P<=0.05, n= 4 in each group.

[0028] FIGS. 8A-8C show tamoxifen mediated Phdl,2,3 ablation. (FIG. 8A) PCR analysis of Phdl from whole retina DNA isolated from 3-week-old retinae identified an 800-900-bp fragment for an untreated mutant and negative control (with no Pde6gCreERT2), and a 481-bp fragment for a mutant treated with tamoxifen at age P7,8, and 10. Lane 1 and 2: Phd'2'', Pde6pH620Q / H620Q,-Pde6gCreERT2with tamoxifen injected at age P7,8,10. Lane 3 and 4: Phd'1'1'. Pde6pH620^ / H620^,'Pde6gCreERT2with sham solution injected at age P7,8,10. Lane 5: negative control, Phd'121'. Pde6pH620Q / H620Q. (FIG. 8B) PCR analysis of Phd2 from whole retina DNA isolated from 3-week-old retinae identified an 840-bp fragment for an untreated mutant and negative control (with no Pde6gCreERT2), and a 140-bp fragment for a mutant treated with tamoxifen at age P7,8 and 10. Lane 1 and 2: Phd '. Pde6pH620Q / H620Q,'Pde6gCreERT2with tamoxifen injected at age P7, 8, 10. Lane 3: Phcf1^,' Pde6 / 3H62(IQ / H62(IQ,'Pde6gCreERT2with sham solution injected at age P7, 8, 10. Lane 4: negative control, Phcf1^1,' Pde6 / 3H62(IQ / H62(l®. (FIG. 8C) PCRanalysis of Phd3 from whole retina DNA isolated from 3-week-old retinae identified an -1000- bp fragment for an untreated mutant and negative control (with no Pde6gCreERT2\ and a -400-bp fragment for a mutant treated with tamoxifen at age P7, 8 and 10. Lane 1 and 2: Phd2,' Pde6 / 3H62(IQ / H62(IQ,-Pde6gCreERT2with tamoxifen injected at age P7, 8, 10. Lane 3: Phcf1^,' Pde6pH620^ / H620^,'Pde6gCreERT2with sham solution injected at age P7, 8, 10. Lane 4: negative control, PhdEp Pde6p"62"Q / "62"Q.

[0029] FIG. 9 shows PHD deficiency decreased mitochondrial pyruvate oxidation. Immunoblotting for regulators of glycolytic metabolism in the retinas of treated and untreated mice at P21 (before the onset of degeneration) revealed increased protein levels of hypoxiainducible factors 1A (HIF1A) and 2A (HIF2A), Phosphofructokinase (PFK1), and Pyruvate Dehydrogenase Kinase 1 (PDK1) in Phd^,' Pde6bH620Q / H620Qmice, p-actin (b-ACTIN) was used as a loading control.

[0030] FIGS. 10A-10D show PHD ablation results in a minimal pentose phosphate pathway activity shift. Retinas were isolated from control or tamoxifen-injected Pd ebb'1620 1620PHDfl / fl; Pde6g-CreERT2mice and incubated in 5 mM l,2-13C2-glucose for 10 minutes. When l,2-13C2-glucose enters the pentose phosphate shunt, it loses the carbon at position 1 in the 6- phosphogluconate dehydrogenase reaction as13CO2. When the remaining carbons re-enter glycolysis as glyceraldehyde-3 -phosphate, they will only carry no13C atoms (m+0) or one13C atom (m+1). Products of l,2-13C2-glucose that skip the pentose phosphate pathway will retain no13C atoms (m+0) or two13C atoms (m+2). Production of m+1 products is an indicator of PPP activity. Metabolites were extracted and derivatized. The abundance of unlabeled (m+0) and13C labeled metabolic intermediates downstream of glucose were determined using gas chromatography-linked mass spectrometry. These intermediates were (FIG. 10A) phosphoenolpyruvate (PEP), (FIG. 10B) pyruvate, (FIG. 10C) lactate, and (FIG. 10D) citrate. Glycolysis is saturated with13C by 10 minutes, yet none of the glycolytic intermediates or downstream products were m+1 labeled by 10 minutes.

[0031] FIGS. 11A and 1 IB show rod photoreceptors are equally preserved in all retina samples. Following metabolite extraction for FIG. 10, leftover protein was extracted in Laemmli sample buffer and 15 pg per sample was run on a 13% polyacrylamide gel via SDS-PAGE. Separated proteins were transferred to a PVDF membrane, which was labeled for recoverin (1 :500, PMID: 1672047) and 13-actin (1:1000, ab8226). (FIG. 10A) The ratio of recoverin / 13-actin wasdetermined using ImageJ vl.54. Samples originating from the same mice were averaged. (FIG. 10B) The recoverin to 13 -actin ratio for all samples was normalized to the mean ratio of recoverin to 13-actin in control samples and compared using an unpaired t-test (p=0.9979; n=6-8).

[0032] FIGS. 12A and 12B show representative ERG single traces. (FIG. 12A) Experimental Pde6p mice ERG traces for data shown in FIG. 4. (FIG. 12B) Representative eight-week-old wild type ERG responses.

[0033] FIG. 13 shows fluorescein angiography of 1 -year old subretinally-transduced RHOCIIOR / +demonstrates normal vascularization. (Left) Infrared fundus image captured to help localize to the center of the fundus. (Right) Fundus fluorescein angiography of subretinally transduced eye. Mouse was subretinally transduced at an age of 1 month and imaged at an age of 1 year old. Normal patterns of vascularization associated with aging and RHOcn0R / +retinal degeneration are observed. Scale bar (white, bottom left) = 200 pM.

[0034] FIGS. 14A-14C show chronic Cas9 expression m Pde6bCas9 / + photoreceptors has no detectable deleterious effects on the maximal ERG response. (FIG. 14A) Cas9 was knocked into the Pde6b locus. (FIG. 14B) The immunoblot shows Cas9 expression in a retinal lysate from a Pde6b-P2A-Cas9 knock-in (KI) mouse (vs. WT, C57BL / 6J). (FIG. 14C) The maximal ERG response of a 6-month-old Cas9 knock-in mouse is indistinguishable from that of a WT C57BL / 6J mouse.

[0035] FIGS. 15A-15C show the on target engagement of the PHD ablation results with AAV8::U6- sgl68; sgl66 PHD2 on targeted photoreceptor neurons. FIG. 15A is the summary of editing events from control mouse retinas the received subretinal delivery of AAV8::U6- sgl68; sgl66 PHD2 , as determined by next generation sequencing of gRNA targeted photoreceptor genomic DNA. FIG. 15B shows exemplary deletions and frameshift mutations induced by sgRNA-166. Sequence analysis of gRNA-targeted photoreceptor genomic DNA reveals editing outcomes, with SEQ ID NOs: 41 to 58 highlighting specific deletions and shifts in the reading frame that disrupt PHD2 function. FIG. 15C shows exemplary deletions and frameshift mutations induced by sgRNA-168. Next-generation sequencing of gRNA-targeted photoreceptor genomic DNA demonstrates distinct mutation profiles, with SEQ ID NOs: 59 to 79 capturing the editing patterns resulting from CRISPR-mediated PHD2 ablation.DETAILED DESCRIPTION

[0036] Retinitis pigmentosa (RP) is one of the most common forms of hereditary neurodegeneration. It is caused by one or more of at least 3100 mutations in over 80 genes that are primarily expressed in rod photoreceptors. In RP, the primary rod-death phase is followed by cone death, regardless of the underlying gene mutation that drove the initial rod degeneration. Dampening the oxidation of glycolytic end-products in rod mitochondria enhances cone survival in divergent etiological disease models independent of the underlying rod-specific gene mutations.

[0037] Therapeutic editing of the prolyl hydroxylase domain-containing protein gene (PHD2, also known as Eglnl) in rod photoreceptors led to the sustained survival of both diseased rods and cones in both preclinical autosomal-recessive and dominant RP models. Adeno-associated virus-mediated CRISPR-based therapeutic reprogramming of the aerobic glycolysis node may serve as a gene-agnostic treatment for patients with various forms of RP. Both pharmacological inhibition with FG-4592 and genetic ablation of PHD 1, 2, 3 (Egln2, 1, 3) increase the ratio of glycolytic to mitochondrial activity and preserve rod and cone survival and function in thePde6PH620&H620Qi^ arRP model. When using a cell-specific therapy, the metabolism of a young healthy retina was preserved in all cell types, allowing more cells which verge on the boundary of death to survive and conveying a thicker ONL and stronger ERG response.

[0038] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.Definitions

[0039] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. As used herein, comprising a certain sequence or a certain SEQ ID NO usually implies that at least one copy of said sequence is present in recited peptide or polynucleotide. However, two or more copies are also contemplated. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0040] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0041] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0042] As used herein, the terms “administering,” “providing,” and “introducing,” are used interchangeably herein and refer to the placement into a subject by a method or route which results in at least partial localization to a desired site. Administration can be by any appropriate route which results in delivery to a desired location in the subject.

[0043] The term “contacting” as used herein refers to bring or put in contact, to be in or come into contact. The term “contact” as used herein refers to a state or condition of touching or of immediate or local proximity.

[0044] The term “gene” refers to a DNA sequence that comprises control and coding sequences necessary for the production of an RNA having a non-coding function (e.g., a ribosomal or transfer RNA), a polypeptide, or a precursor of any of the foregoing. The RNA or polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained. Thus, a “gene” refers to a DNA or RNA, or portion thereof, that encodes a polypeptide or an RNA chain that has functional role to play in an organism. For the purpose of this disclosure, it may be considered that genes include regions that regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions.

[0045] As used herein, a “nucleic acid” or a “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and / or purine bases, preferably cytosine, thymine, and uracil, andadenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793- 800 (Worth Pub. 1982)). The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. In some embodiments, a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA / RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002)) and U.S. Pat. No. 5,034,506), locked nucleic acid (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc., 122: 8595-8602 (2000)), and / or a ribozyme. Hence, the term “nucleic acid” or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and / or non- nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or doublestranded, and represent the sense or antisense strand. The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0046] As used herein, the term “preventing” refers to partially or completely delaying onset of a disease, disorder and / or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular disease, disorder, and / or condition; partially or completely delaying progression from a particular disease, disorder and / or condition; and / or decreasing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0047] As used herein, “treat,” “treating,” and the like means a slowing, stopping, or reversing of progression of a disease or disorder. The term also means a reversing of theprogression of such a disease or disorder. As such, “treating” means an application or administration of the methods or devices described herein to a subject, where the subject has a disease or a symptom of a disease, where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease or symptoms of the disease.

[0048] A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non- human) that may benefit from the administration of devices and systems contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment of the methods herein, the mammal is a human.

[0049] A “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, e.g., an “insert,” may be attached or incorporated so as to bring about the replication of the attached segment in a cell.

[0050] The term “wild-type” refers to a gene or a gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wild-type” form of the gene. In contrast, the term “modified,” “mutant,” or “polymorphic” refers to a gene or gene product that displays modifications in sequence and or functional properties (e.g., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.

[0051] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.Methods

[0052] Disclosed herein are components, systems, and methods which edit or ablate a prolyl hydroxylase domain-containing protein (PHD) gene. There are at least three different PHD isoforms, PHD1, PHD2, and PHD3, also referred to as EGLN2, EGLN1, and EGLN3, or HPH3, HPH2, and HPH1, respectively. In select embodiments, the disclosed components, systems, and methods edit or ablate PHD2.

[0053] The gene editing or ablation may comprise full ablation of the target gene (e.g., PHD2), truncation of the target gene (e.g., PHD2), or any edit which decreases the target gene (e.g., PHD2) or protein expression. The editing or ablation may comprise use of any agent and / or composition that decreases the level and / or activity of PHD2. Exemplary agents and / or compositions also include, without limitation, gene editing systems (e.g., a CRISPR-Cas system), siRNA, shRNA, miRNA, antisense oligonucleotides, and / or a chemical / small molecules.

[0054] In some embodiments, the methods comprise contacting a prolyl hydroxylase domaincontaining protein 2 (PHD2) gene with a gene editing system which specifically targets PHD2. The gene editing system may be used to modulate (e.g., repress) PHD2, introduce one or more nucleotide substitutions, addition, or deletions into PHD2 (e.g., to insert a mutation, disrupt target gene expression, or alter a promoter region for a target gene), truncate PHD2, or delete PHD2. For example, the gene editing system may comprise a zinc-finger nuclease, a homing endonuclease, a TALEN (transcription activator-like effector nuclease), a NgAgo (argonaute endonuclease), a SGN (structure-guided endonuclease), or an RNA-guided endonuclease, e.g., a CRISPR-Cas system. Accordingly, the gene editing system may be a site directed gene editing system, such as a site-specific recombination-based system, zinc finger nuclease (ZFN)- or transcription activator-like effector nucleases (TALEN)-mediated gene editing system, or a CRISPR / Cas gene editing system. The gene editing system may comprise one or more Cas proteins (e.g., Cas9), or other RNA-guided nucleases, to work in conjunction with one or more guide RNAs (gRNAs) directed to the target gene (e.g., PHD2). In select embodiments, the gene editing system is a CRISPR / Cas based system comprising one or more Cas proteins and one or more gRNAs.

[0055] A “CRISPR-Cas system” refers collectively to transcripts and other elements involved in the expression of and / or directing the activity of CRISPR-associated (“Cas”) genes, includingsequences encoding a Cas gene, Cas protein, a cr (CRISPR) sequence (e.g., crRNA or an active partial crRNA), or other sequences and transcripts from a CRISPR locus. CRISPR-Cas editing technology is described in detail in, for example, U.S. Patent Nos. 8,546,553, 8,697,359; 8,771,945; 8,795,965; 8,865,406; 8,871,445; 8,889,356; 8,889,418; 8,895,308; 8,9066,616; 8,932,814; 8,945,839; 8,993,233; 8,999,641; 9,115,348; 9,149,049; 9,493,844; 9,567,603; 9,637,739; 9,663,782; 9,404,098; 9,885,026; 9,951,342; 10,087,431; 10,227,610; 10,266,850; 10,601,748; 10,604,771; and 10,760,064; and U.S. Patent Application Publication Nos.US2010 / 0076057; US2014 / 0113376; US2015 / 0050699; US2015 / 0031134; US2014 / 0357530; US2014 / 0349400; US2014 / 0315985; US2014 / 0310830; US2014 / 0310828; US2014 / 0309487; US2014 / 0294773; US2014 / 0287938; US2014 / 0273230; US2014 / 0242699; US2014 / 0242664; US2014 / 0212869; US2014 / 0201857; US2014 / 0199767; US2014 / 0189896; US2014 / 0186919; US2014 / 0186843; and US2014 / 0179770, each incorporated herein by reference. The CRISPR / Cas system may be derived from any type of CRISPR system, e.g., a CRISPR type II system or a CRISPR type V system.

[0056] Cas protein families are described in detail in the art, e.g., Haft et al., PLoS Comput. Biol., 1(6): e60 (2005), incorporated herein by reference. The Cas protein may be any Cas endonucleases. The Cas nuclease can be a wild-type Cas nuclease or a modified Cas nuclease, or a fragment thereof. The Cas nuclease can be modified to increase nucleic acid binding affinity and / or specificity, alter an enzymatic activity, and / or change another property of the protein.

[0057] In some embodiments, the Cas protein is Cas9 or Cas 12a, otherwise referred to as Cpfl , or a modified variant thereof. In one embodiment, the Cas9 protein is a wild-type Cas9 protein. The Cas9 protein can be obtained from any suitable microorganism, and a number of bacteria express Cas9 protein orthologs or variants. In some embodiments, the Cas9 is from Streptococcus pyogenes or Staphylococcus aureus. Cas9 proteins of other species are known in the art (see, e.g., U.S. Patent Application Publication 2017 / 0051312, incorporated herein by reference) and may be used in connection with the present invention. The amino acid sequences of Cas proteins from a variety of species are publicly available through the GenBank and UniProt databases.

[0058] In some embodiments, the one or more gRNAs are configured to target PHD2. In some embodiments, the one or more gRNAs are each configured to bind and cleave exon 1 of PHD2. In select embodiments, the gene editing system comprises two or more gRNAs, eachconfigured to target, e.g., bind and / or cleave, exon 1 of PHD2. In select embodiments, the gene editing system comprises two gRNAs having nucleotide sequences of SEQ ID NOs: 11 and 12.

[0059] Accordingly, in some aspects disclosed herein are gene editing systems comprising one or more RNA-guided nucleases and one or more guide RNAs (gRNAs) directed to target PHD2. In select embodiments, disclosed herein are gene editing systems comprising one or more Cas nucleases and one or more guide RNAs (gRNAs) directed to target PHD2. In some embodiments, the gene editing systems comprise two or more guide RNAs, each configured to target exon 1 of PHD2. In some embodiments, the gene editing systems comprise two or more guide RNAs, each configured to target different sites in exon 1 of PHD2. In some embodiments, the gene editing systems comprise Cas9. In select embodiments, the gene editing system comprises two gRNAs having nucleotide sequences of SEQ ID NOs: 11 and 12.

[0060] In some embodiments, the one or more guide RNAs are encoded in a CRISPR RNA (crRNA) array. CRISPR arrays contain a series of direct repeats separated by short sequences called spacers. The CRISPR RNA (crRNA) may contain multiple copies of a single gRNA or may contain more than one gRNA wherein each configured to hybridize a distinct target sequence.

[0061] The gRNA may be a crRNA, crRNA / tracrRNA (or single guide RNA, sgRNA). The terms “guide RNA,” “single guide RNA,” and “synthetic guide RNA,” are used interchangeably herein and may refer to a nucleic acid sequence comprising a guide sequence. The terms “guide sequence,” “guide,” and “spacer,” are used interchangeably herein and refer to the nucleotide sequence within a guide RNA that specifies the target gene.

[0062] In addition to a guide sequence, in some embodiments, the gRNA may also comprise a scaffold sequence (e.g., tracrRNA). In some embodiments, such a chimeric gRNA may be referred to as a single guide RNA (sgRNA). Exemplary scaffold sequences will be evident to one of skill in the art and can be found, for example, in Jinek, et al. Science (2012) 337(6096):816- 821, and Ran, et al. Nature Protocols (2013) 8:2281-2308, incorporated herein by reference in their entireties.

[0063] In some embodiments, the gRNA sequence does not comprise a scaffold sequence and a scaffold sequence is expressed as a separate transcript. In such embodiments, the gRNAsequence further comprises an additional sequence that is complementary to a portion of the scaffold sequence and functions to bind (hybridize) the scaffold sequence.

[0064] The gRNA or guide sequence may be between 15-40 nucleotides in length. In some embodiments, the guide sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. gRNAs or sgRNA(s) used in the present disclosure can be between about 5 and 100 nucleotides long, or longer (e.g., 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 60, 61,62, 63, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86,87, 88, 89, 90, 91 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length, or longer).

[0065] “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule, which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization. In some embodiments, the gRNA sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary to the target sequence (e.g., regions of exon 1 of PHD2).

[0066] The gRNA may be a non-naturally occurring gRNA. The gRNA may contain one or more chemically modified nucleotides (e.g., modifications in the ribose group, phosphate group, nucleobase, backbone, or any combination thereof).

[0067] Methods for Conserving Retinal Cells

[0068] In one aspect disclosed herein are methods for increasing retinal cell survival and / or preserving retinal function. In some embodiments, the methods comprise contacting a retinal cell with one or more components, systems, or compositions which edit or ablate a prolyl hydroxylase domain-containing protein (PHD) gene. In some embodiments, the methods comprise contacting a retinal cell with one or more components, systems, or compositions which edit or ablate PHD2. In some embodiments, the methods decrease expression and / or truncate the PHD gene, e.g., PHD2 gene.

[0069] In some embodiments, the methods comprise contacting a retinal cell with one or more gene editing systems which edit or ablate a prolyl hydroxylase domain- containing protein (PHD)gene. In select embodiments, the methods comprise contacting a retinal cell with one or more gene editing systems which edit or ablate a prolyl hydroxylase domain-containing protein 2 (PHD2) gene. The gene editing systems disclosed above are applicable to the methods for increasing retinal cell survival and / or preserving retinal function.

[0070] In some embodiments, the gene editing system is a CRISPR / Cas gene editing system. As described above, the gene editing system may comprise one or more Cas proteins (e.g., Cas9), or other RNA-guided nucleases and one or more guide RNAs (gRNAs) directed to the target gene (e.g., PHD2). In some embodiments, the gene editing systems comprise two or more guide RNAs, each configured to target exon 1 of PHD2. In some embodiments, the gene editing systems comprise two or more guide RNAs, each configured to target different sites in exon 1 of PHD2. In some embodiments, the gene editing systems comprise Cas9. In select embodiments, the gene editing system comprises two gRNAs having nucleotide sequences of SEQ ID NOs: 11 and 12.

[0071] The retinal cell may be any cell type or class of cells from the retina. For example, the retinal cell may include, but is not limited to, photoreceptor cells (e.g., rods; cones), retinal ganglion cells (RGC), glial cells (e.g., a Muller glial cell, a microglial cell), bipolar cells, amacrine cells, horizontal cells, and / or retinal pigmented epithelium (RPE) cells. In some embodiments, the retinal cell is a photoreceptor cell. In select embodiments, the retinal cell is a rod cell. In select embodiments, the retinal cell is a cone cell.

[0072] In some embodiments, the retinal cell(s) is in a subject. In such embodiments, the methods comprise administering one or more components, systems, or compositions which edit or ablate a prolyl hydroxylase domain-containing protein (PHD) gene to the subject. In some embodiments, the methods comprise administering one or more components, systems, or compositions which edit or ablate PHD2 to the subject. In some embodiments, the methods decrease expression and / or truncate the PHD gene, e.g., PHD2 gene, in the subject.

[0073] Methods of treating a disease or disorder

[0074] Also disclosed herein are methods for treating or preventing a disease or disorder in a subject. In some embodiments, the methods comprise administering one or more components, systems, or compositions which edit or ablate a prolyl hydroxylase domain-containing protein (PHD) gene to the subject. In some embodiments, the methods comprise administering one or more components, systems, or compositions which edit or ablate PHD2 to the subject. In someembodiments, the methods decrease expression and / or truncate the PHD gene, e.g., PHD2 gene, in the subject.

[0075] In some embodiments, the methods comprise contacting a retinal cell with one or more gene editing systems which edit or ablate a prolyl hydroxylase domain-containing protein (PHD) gene. In select embodiments, the methods comprise contacting a retinal cell with one or more gene editing systems which edit or ablate a prolyl hydroxylase domain-containing protein 2 (PHD2) gene. The gene editing systems disclosed above are applicable to the methods for increasing retinal cell survival and / or preserving retinal function.

[0076] In some embodiments, the disease or disorder comprises one or more retinal degenerative diseases or disorders. Retinal degenerative disease or disorders suitable for treatment with the disclosed methods include, but are not limited to retinitis pigmentosa (RP), age-related macular degeneration (AMD), glaucoma, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and / or Lewy body dementia. In select embodiments, the retinal degenerative disease or disorder is retinitis pigmentosa (RP).

[0077] In some embodiments, the systems, or components thereof, for editing or ablating a prolyl hydroxylase domain-containing protein (PHD) gene are configured for delivery to retinal cells. In some embodiments, the system is configured for delivery to rod and cone photoreceptor cells. For example, in some embodiments, the nucleic acids encoding the components may comprise a retinal cell (e.g., rod and / or cone photoreceptor cell) promoter which directs expression of the components in the retinal cells. In some embodiments, mini promoters, minimal promoter element(s) designed for expression in specific types (e.g., rod and / or cone photoreceptor cell) are used.

[0078] Suitable retinal, rod, and / or cone photoreceptor cell promoters include, but are not limited to: 770En_454P(hGRAfb), a human GRM6 gene-derived, short promoter; promoters based on the 2.1 -kb human L-opsin promoter (pR2.1); promoter derived from the rhodopsin kinase (RK) gene; promoter derived from the rhodopsin gene; a promoter derived from the Nrl gene; murine rhodopsin promoter (mOP); G-protein-coupled receptor protein kinase 1 (GRK1) promoter; retinol-binding protein 3, interstitial (RBP3) promoter; RPE65 promoter; human inter-photoreceptor retinoid binding protein / retinol-binding protein 3 (IRBP) promoter; and retinaldehyde binding protein 1 (RLBP1) promoter. Suitable photoreceptor cell promoters include, but are not limited to: interphotoreceptor retinoid-binding protein (IRBP), cone arrestin(CAR), rhodopsin (RHO), PR1.7 (a truncated version of version of the L-opsin promoter), synthetic promoters: ProAl, ProA6, ProCi, ProA14, and ProA36, and G protein-coupled receptor kinase 1 (GRK1).

[0079] Additionally, or alternatively, the systems, or components thereof, for editing or ablating a prolyl hydroxylase domain-containing protein (PHD) gene are configured for administration to the eye and / or retina, rather than systemic administration.

[0080] Administration may be through any suitable mode of administration, including but not limited to: intravenous, intra-arterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intracerebroventricular, sub-retinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmucosal, topical, and inhalation. In some embodiments, the systems or components are delivered to the tissue(s) of interest (e.g., the retina). In select embodiments, the administration is by intravitreal or subretinal injection. Delivery may be either via a single dose, or multiple doses.

[0081] In some embodiments, an effective amount of the components of the systems, or components thereof, for editing or ablating a prolyl hydroxylase domain-containing protein (PHD) gene as described can be administered. As used herein the term “effective amount” may be used interchangeably with the term “therapeutically effective amount” and refers to that quantity that is sufficient to result in a desired activity upon administration to a subject in need thereof. Within the context of the present disclosure, the term “effective amount” refers to that quantity of the components of the system such that successful editing and ablating of the target PHD gene (e.g., PHD2) is achieved

[0082] When utilized as a method of treatment, the effective amount may depend on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. In some embodiments, the effective amount alleviates, relieves, ameliorates, improves, reduces the symptoms, or delays the progression of any disease or disorder in the subject. In some embodiments, the subject is a human.1. Nucleic Acids and Delivery

[0083] The present disclosure also provides for one or more nucleic acids encoding agents and / or compositions that decrease the level and / or activity of PHD, e.g., the gene editing systems or components thereof as disclosed herein for editing or ablating a prolyl hydroxylase domaincontaining protein (PHD) gene. In some embodiments, the one or more nucleic acids comprise one or more messenger RNAs, one or more vectors, or any combination thereof.

[0084] The present disclosure provides one or more vectors encoding the gene editing systems, or components thereof, for editing or ablating a prolyl hydroxylase domain-containing protein (PHD) gene and cells containing the vector(s), thereof. The vector(s) may be used to propagate the nucleic acid in an appropriate cell and / or to allow expression from the nucleic acid (e.g., an expression vector). The person of ordinary skill in the art would be aware of the various vectors available for propagation and expression of a nucleic acid sequence.

[0085] In certain embodiments, vectors of the present disclosure can drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, Nature (1987) 329:840, incorporated herein by reference) and pMT2PC (Kaufman, et al., EMBO J. (1987) 6:187, incorporated herein by reference). When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd eds., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference.

[0086] The vectors of the present disclosure may direct the expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Such regulatory elements include promoters that may be tissue specific or cell specific. The term “tissue specific” as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., seeds) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue. The term “cell type specific” as applied to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in adifferent type of cell within the same tissue. The term “cell type specific” when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., immunohistochemical staining. In some embodiments, the promoter is specific to retinal cells. In some embodiments, the promoter directs expression in rod and / or cone photoreceptor cells.

[0087] Nucleic acids of the present disclosure can comprise any of a number of promoters known to the art, wherein the promoter is constitutive, regulatable or inducible, cell type specific, tissue-specific, or species specific. In addition to the sequence sufficient to direct transcription, a promoter sequence of the invention can also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, Kozak sequences and introns). Many promoter / regulatory sequences useful for driving constitutive expression of a gene are available in the art and include, but are not limited to, for example, CMV (cytomegalovirus promoter), EFla (human elongation factor 1 alpha promoter), SV40 (simian vacuolating virus 40 promoter), PGK (mammalian phosphoglycerate kinase promoter), Ubc (human ubiquitin C promoter), human beta-actin promoter, rodent beta-actin promoter, CBh (chicken beta-actin promoter), CAG (hybrid promoter contains CMV enhancer, chicken beta actin promoter, and rabbit betaglobin splice acceptor), TRE (Tetracycline response element promoter), Hl (human polymerase III RNA promoter), U6 (human U6 small nuclear promoter), and the like. Additional promoters that can be used for expression of the components of the present system, include, without limitation, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Maloney murine leukemia virus (MMLV) LTR, myeoloproliferative sarcoma virus (MPSV) LTR, spleen focus-forming virus (SFFV) LTR, the simian virus 40 (SV40) early promoter, herpes simplex tk virus promoter, elongation factor 1- alpha (EFl -a) promoter with or without the EFl -a intron. Additional promoters include any constitutively active promoter. Alternatively, any regulatable promoter may be used, such that its expression can be modulated within a cell.

[0088] Moreover, inducible expression can be accomplished by placing the nucleic acid encoding such a molecule under the control of an inducible promoter / regulatory sequence. Promoters that are well known in the art can be induced in response to inducing agents such as metals, glucocorticoids, tetracycline, hormones, and the like, are also contemplated for use withthe invention. Thus, it will be appreciated that the present disclosure includes the use of any promoter / regulatory sequence known in the art that is capable of driving expression of the desired protein operably linked thereto.

[0089] In some embodiments, the one or more vectors are configured for delivery to retinal cells. In some embodiments, the one or more vectors are configured for delivery to rod and cone photoreceptor cells. For example, in some embodiments, the one or more vectors may comprise a retinal cell (e.g., rod and / or cone photoreceptor cell) promoter which directs expression of the components in the retinal cells. In some embodiments, mini promoters, minimal promoter element(s) designed for expression in specific types (e.g., rod and / or cone photoreceptor cell are used.

[0090] Suitable retinal, rod, and / or cone photoreceptor cell promoters include, but are not limited to: 770En_454P(liG7? / V / 6), a human GRM6 gene-derived, short promoter; promoters based on the 2.1 -kb human L-opsin promoter (pR2.1); promoter derived from the rhodopsin kinase (RK) gene; promoter derived from the rhodopsin gene; a promoter derived from the Nrl gene; murine rhodopsin promoter (mOP); G-protein-coupled receptor protein kinase 1 (GRK1) promoter; retinol-binding protein 3, interstitial (RBP3) promoter; RPE65 promoter; human inter-photoreceptor retinoid binding protein / retinol-binding protein 3 (IRBP) promoter; and retinaldehyde binding protein 1 (RLBP1) promoter. Suitable photoreceptor cell promoters include, but are not limited to: interphotoreceptor retinoid-binding protein (IRBP), cone arrestin (CAR), rhodopsin (RHO), PR1.7 (a truncated version of version of the L-opsin promoter), synthetic promoters: ProAl, ProA6, ProCi, ProA14, and ProA36, and G protein-coupled receptor kinase 1 (GRK1).

[0091] Additionally, or alternatively, the one or more vectors are configured for administration to the eye and / or retina, rather than systemic administration.

[0092] Additionally, the vector may contain, for example, some or all of the following: a selectable marker gene for selection of stable or transient transfectants in host cells; transcription termination and RNA processing signals; 5’-and 3 ’-untranslated regions; internal ribosome binding sites (IRESes), versatile multiple cloning sites; and reporter gene for assessing expression of the chimeric receptor. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art. Selectable markers include chloramphenicol resistance, tetracycline resistance, spectinomycin resistance, neomycin,streptomycin resistance, erythromycin resistance, rifampicin resistance, bleomycin resistance, thermally adapted kanamycin resistance, gentamycin resistance, hygromycin resistance, trimethoprim resistance, dihydro folate reductase (DHFR), GPT; the URA3, HIS4, LEU2, and TRP1 genes of S. cerevisiae.

[0093] When introduced into a cell, the vector may be maintained as an autonomously replicating sequence or extrachromosomal element or may be integrated into host DNA.

[0094] Viral and non-viral based gene transfer methods can be used to introduce the nucleic acids into cells, tissues, or a subject. Such methods can be used to administer the nucleic acids to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, cosmids, RNA (e.g., a transcript of a vector described herein), a nucleic acid, and a nucleic acid complexed with a delivery vehicle.

[0095] Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. A variety of viral constructs may be used to deliver the present nucleic acids to the cells, tissues and / or a subject. Viral vectors include, for example, retroviral, lentiviral, adenoviral, adeno-associated, baculoviral, and herpes simplex viral vectors. Nonlimiting examples of such recombinant viruses include recombinant adeno-associated virus (AAV), recombinant adenoviruses, recombinant lentiviruses, recombinant retroviruses, recombinant herpes simplex viruses, recombinant baculoviruses, recombinant poxviruses, phages, etc. The present disclosure provides vectors capable of integration in the host genome, such as retrovirus or lentivirus. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989; Kay, M. A., et al., 2001 Nat. Medic. 7( 1): 33-40; and Walther W. and Stein U., 2000 Drugs, 60(2): 249-71, incorporated herein by reference.

[0096] In one embodiment, the vector or vectors are derived from or based on adeno- associated viruses (AAVs). Adeno-associated viruses (AAV), from the parvovirus family, are small viruses with a genome of single stranded DNA. Because AAV are not associated with pathogenic disease in humans, AAV vectors are able to deliver therapeutic proteins and agents to human patients without causing substantial AAV pathogenesis. The adeno-associated virus may be of any serotype, a mixture of serotypes, or variants thereof. Exemplary AAV serotypes include AAV1, AAV 2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV1 1. For example, when the viral transfer vector is based on a mixture of serotypes, the viral transfer vector may contain the capsid signal sequences taken from one AAV serotype (forexample selected from any one of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11) and packaging sequences from a different serotype (for example selected from any one of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11). In select embodiments, the vector or vectors are derived from or based on AAV8.

[0097] An AAV vector, as used herein, is a vector which comprises at least one component part derivable from adeno-associated viruses. That component part may be involved in the biological mechanisms by which the vector infects cells, expresses genes, or is replicated. In some embodiments, all or a part of the viral genome has been replaced with a transgene, which is a non-native nucleic acid with respect to the AAV nucleic acid sequence. AAV vectors generally have had up to approximately 96% of the parental genome deleted, such that only the terminal repeats (ITRs), which contain recognition signals for DNA replication and packaging, remain. Thus, the AAV vector may be a recombinant AAV vector.

[0098] For a description of AAV-based vectors, see, for example, U.S. Pat. Nos. 8,679,837,8,637,255, 8,409,842, 7,803,622, and 7,790,449, and U.S. Publication Nos. 20150065562, 20140155469, 20140037585, 20130096182, 20120100606, and 20070036757, incorporated herein by reference in their entirety.

[0099] In some embodiments, the vector(s) may be configured or modified to confer increased infectivity of one or more types of cells. In the case of two or more vectors, each vector may be configured to confer increased infectivity in the same or different cell types. In some embodiments, the vector(s) may be configured to confer increased infectivity in one or more types of retinal cells (e.g., a photoreceptor cell (e.g., rods; cones), a retinal ganglion cell (RGC), a glial cell (e.g., a Muller glial cell, a microglial cell), a bipolar cell, an amacrine cell, a horizontal cell, and / or a retinal pigmented epithelium (RPE) cell). See for example, International Patent Publication No. WO2019104279, incorporated herein by reference in its entirety.

[0100] Due to size constraints of viral genomes for packaging, the systems, or components thereof, for editing or ablating a prolyl hydroxylase domain-containing protein (PHD) gene can be engineered and packaged in two or more vectors / stocks. Whether packaged in one vector or stock which is used as a composition according to the invention, or in two or more vectors or stocks which form a virus composition of the invention, the composition collectively contains systems, or components thereof, for editing or ablating a prolyl hydroxylase domain-containing protein (PHD) gene.

[0101] Vectors according to the present disclosure can be transformed, transfected, or otherwise introduced into a wide variety of host cells. Transfection refers to the taking up of a vector by a cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, lipofectamine, calcium phosphate co-precipitation, electroporation, DEAE-dextran treatment, microinjection, viral infection, and other methods known in the art. Transduction refers to entry of a virus into the cell and expression (e.g., transcription and / or translation) of sequences delivered by the viral vector genome. In the case of a recombinant vector, “transduction” generally refers to entry of the recombinant viral vector into the cell and expression of a nucleic acid of interest delivered by the vector genome.

[0102] Methods of delivering vectors to cells may include DNA or RNA electroporation, transfection reagents such as liposomes or nanoparticles to delivery DNA or RNA; delivery of DNA, RNA, or protein by mechanical deformation (see, e.g., Sharei et al. Proc. Natl. Acad. Sci. USA (2013) 110(6): 2082-2087, incorporated herein by reference); or viral transduction. In some embodiments, the vectors are delivered to host cells by viral transduction. Nucleic acids can be delivered as part of a larger construct, such as a plasmid or viral vector, or directly, e.g., by electroporation, lipid vesicles, viral transporters, microinjection, and biolistics (high-speed particle bombardment). Similarly, the nucleic acids or vectors can be delivered by any method appropriate for introducing nucleic acids into a cell. In some embodiments, the construct or the nucleic acid encoding the systems, or components thereof, for editing or ablating a prolyl hydroxylase domain-containing protein (PHD) gene is a DNA molecule. In some embodiments, the nucleic acid encoding the systems, or components thereof, for editing or ablating a prolyl hydroxylase domain-containing protein (PHD) gene is a DNA vector and may be electroporated to cells. In some embodiments, the nucleic acid encoding the systems, or components thereof, for editing or ablating a prolyl hydroxylase domain-containing protein (PHD) gene is an RNA molecule, which may be electroporated to cells.

[0103] Additionally, delivery vehicles such as nanoparticle- and lipid-based delivery systems can be used. Further examples of delivery vehicles include lentiviral vectors, ribonucleoprotein (RNP) complexes, lipid-based delivery system, gene gun, hydrodynamic, electroporation or nucleofection microinjection, and biolistics. Various gene delivery methods are discussed indetail by Nayerossadat et al. (Adv Biomed Res. 2012; 1: 27) and Ibraheem et al. (Int J Pharm. 2014 Jan 1 ;459(1 -2):70-83), incorporated herein by reference.

[0104] As such, the disclosure provides an isolated cell comprising the vector(s) or nucleic acid(s) disclosed herein. Preferred cells are those that can be easily and reliably grown, have reasonably fast growth rates, have well characterized expression systems, and can be transformed or transfected easily and efficiently. Examples of suitable prokaryotic cells include, but are not limited to, cells from the genera Bacillus (such as Bacillus subtilis and Bacillus brevis), Escherichia (such as E. coli), Pseudomonas, Streptomyces , Salmonella, and Envinia. Suitable eukaryotic cells are known in the art and include, for example, yeast cells, insect cells, and mammalian cells. Examples of suitable yeast cells include those from the genera Kluyveromyces , Pichia, Rhino-sporidium, Saccharomyces , and Schizosaccharomyces . Exemplary insect cells include Sf-9 and HIS (Invitrogen, Carlsbad, Calif.) and are described in, for example, Kitts et al., Biotechniques, 14'. 810-817 (1993); Lucklow, Curr. Opin. Biotechnol., 4'. 564-572 (1993); and Lucklow et al., J. Virol., 67: 4566-4579 (1993), incorporated herein by reference. A number of suitable mammalian and human host cells are known in the art, and many are available from the American Type Culture Collection (ATCC, Manassas, Va.). Examples of suitable mammalian cells include, but are not limited to, Chinese hamster ovary cells (CHO) (ATCC No. CCL61), CHO DHFR-cells (Urlaub et al., Proc. Natl. Acad. Sci. USA, 97: 4216-4220 (1980)), human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), and 3T3 cells (ATCC No. CCL92). Other suitable mammalian cell lines are the monkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651), as well as the CV-1 cell line (ATCC No. CCL70).Further exemplary mammalian host cells include primate, rodent, and human cell lines, including transformed cell lines. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants, are also suitable. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, HEK, A549, HepG2, mouse L- 929 cells, and BHK or HaK hamster cell lines. Methods for selecting suitable mammalian cells and methods for transformation, culture, amplification, screening, and purification of cells are known in the art.

[0105] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cellis in vitro. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vivo and delivery to the cell comprises administration to a subject.2. Compositions

[0106] The agents that decrease the level and / or activity of PHD, e.g., the gene editing systems or components thereof as disclosed herein, for editing or ablating a prolyl hydroxylase domain-containing protein (PHD) gene may be provided in a composition. For example, the gene editing systems, or components thereof, for editing or ablating a prolyl hydroxylase domaincontaining protein (PHD) gene, or nucleic acids or vectors encoding thereof, are provided as a composition including additional excipients or pharmaceutically acceptable carriers. The choice of excipients or pharmaceutically acceptable carriers will depend on factors including, but not limited to, the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

[0107] Excipients and carriers may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. Some examples of materials which can serve as excipients and / or carriers are sugars including, but not limited to, lactose, glucose and sucrose; starches including, but not limited to, com starch and potato starch; cellulose and its derivatives including, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients including, but not limited to, cocoa butter and suppository waxes; oils including, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols; including propylene glycol; esters including, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents including, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants including, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants. The compositions of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Techniques and formulations may be found, for example, in Remington’s Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).EXAMPLESMaterials and Methods

[0108] Preclinical Models Pde6 / 3H620Q / H620Q:, Pde6yCreERT2l+is a well-established preclinical model of arRP; Rhocll0R / +is a well-established preclinical model of adRP. Pde6yCreERT2l+mice were generated at Jonas Children's Vision Care laboratory; PHD 1,2, 3 mouse model PHD 1,2, 3^ mice [Egln2tm2Fon8'Egln ltm2Fon8Egln3tm2Fon8'l3} were obtained from Jackson Laboratory (stock no. 028097). The resulting progeny were bred with PHD!1,2^ ,3? mice. Pde6 / )'1620- '1620~ and Pde6yCreERT2mice used herein were rederived using previously published methods (Zhang L, et al. Hum Mol Genet. 2016;25(19):4244-55). Pde6[)F62IEFH62l'(2and Pde6yCreERT2lines were crossed to yield experimental lines in this research study.

[0109] All mice were housed in the Edward S. Harkness Eye Institute at Columbia University Irving Medical Center Facility under a 12-hour light and 12-hour dark cycle. When applicable, mice were euthanized following the Columbia University IACUC guidelines and using previously reported and approved methods. Mice were used in accordance with the Statement for the Use of Animals in Ophthalmic and Vision Research of the Association for Research in Vision and Ophthalmology and the Policy on the Use of Animals in Neuroscience Research of the Society for Neuroscience.

[0110] PHD inhibitor 5 mg FG-4592 (M.C.E.; HY-13426) was dissolved in 100 pl DMSO (Sigma-Aldrich; 67-68-5) at 42 °C to generate a stock solution and subsequently mixed at a ratio of 1 :9 in Sunflower oil (Sigma-Aldrich; 8001-21-6). 20 mg / kg BW of FG-4592 was administered via oral gavage to the Pde6 / 3H620Q / H620Q535 mouse model as treatment group. The same volume of DMSO was mixed 1:9 with sunflower oil and fed to the retinal degenerating mice for the control group.

[0111] Histology Experimental and control retinae were harvested from euthanized mice to observe retinal morphology, and retinae were stained with H&E. The H&E-stained samples were used to quantify the thickness of the retinal ONL. ONL layers are labeled with a yellow or green bar in figures within this manuscript.

[0112] Electroretinography ERG tests were administered to both eyes of all mice. ERG testing was performed 6-, 8-, and 10-weeks post-injection. Mice were dark-adapted for 12 hours prior to anesthetization with 0.1 ml / 10 g B.W. of 1 ml of 100 mg / ml ketamine and 0.1 ml of 20 mg / ml xylazine in 8.9 ml PBS, which was injected i.p. During anesthetization, mice were placedon heating pads to maintain their body temperature at 37°C. Mouse eyes were dilated with one drop per eye of Tropicamide Ophthalmic Solution (1%; Akom). Electrodes were placed on the corneas, and Gonak Hypromellose Ophthalmic Demulcent Solution (2.5%; Akom) was applied to the eyes to prevent corneal scarring.

[0113] ERG recordings were simultaneous for both eyes. Pulses of 0.00130 cd / m2 and 3 cd / m2 (White-6500K) were employed. This allowed the specific testing of rod and cone functionality so that the individual and combined function could be analyzed.

[0114] Fluorescein Angiography (FA) FA was conducted at 1-year post subretinal AAV transduction of dual gRNA and Cas9 plasmids. Mice were anesthetized with 0.1 ml / 10 g B.W. of 1 ml of 100 mg / ml ketamine and 0.1 ml of 20 mg / ml xylazine in 8.9 ml PBS, injected i.p. Following anesthesia, Tropicamide Opthlamic Solution (1%; Akom) was used to dilate pupils. After proper dilation, 100 pL of Fluorescein Sodium (25%; Akom) was injected i.p. and a Heidelberg Spectralis machine was used to capture angiographic images in the infrared and FA channels.

[0115] Tamoxifen injection and DNA recombination Assay Tamoxifen (Sigma- Aldrich; T5648) was injected intraperitoneally (i.p.) 3 times at a concentration of 100 pg / g B.W. as the treatment group. For the control group, a sham injection was administered with 10% Ethanol (w / w) in sunflower oil (Sigma-Aldrich; S5007). Mice were injected at P7 and tested 4-, 6-, 8-, 10-, and 12-weeks post-injection.

[0116] To assay for recombination, DNA was extracted from the whole retina by using a DNeasy Blood & Tissue Kit (Qiagen, #69506). PCR was performed as previously described (Takeda K, et al., Mol Cell Biol. 2006;26(22):8336-46). Primers that target Eglnl, Egln2, and Egln3 segments of DNA are listed in the provided Table. The protein was extracted from the whole retina to assay for successful recombination. Immunoblots were performed to compare the protein levels of PHD1, PHD2, and PHD3 between treatment and control groups.

[0117] Mouse retinae collection Retinae were harvested from mice at P21 -P23 and snap- frozen in liquid nitrogen before being stored at -80 °C for further processing; DNA, RNA, protein, or mass spectrometry.

[0118] RNA extraction and qRT-PCR Total RNA was extracted from retinae or cell lines using an RNeasy mini kit (QIAGEN, #74104) and was reverse transcribed using SuperScript III First-Strand Synthesis SuperMix (ThermoFisher, 18080-400). The reactions were run aspreviously described (Takeda K, et al., Mol Cell Biol. 2006;26(22):8336-46). Transcript levels of each target gene were determined by SYBR Green-based qPCR (BIO-RAD, 1725271) and were standardized to p-Actin.

[0119] Immunoblotting of retinal lysates At postnatal week 3 (P21) before the onset of ONL loss, retinae were harvested and prepared for immunoblot using previously described experimental procedures (Zhang L, et al. J Clin Invest. 2016;126(12):4659-73). The protein lysate was resuspended, and the supernatant was collected and subject to SDS-polyacrylamide gel electrophoresis using 4 - 15% BIO-RAD TGX pre-cast gels (#4561083). The proteins were transferred to nitrocellulose membranes for western blotting analysis. Whole-cell proteins were normalized to p-Actin (Cell Signaling, #3700). The membrane was stripped and re-probed for all targets (singular membrane). Immunoblotting signals were visualized by an iBright FL 1500 Imaging System (ThermoFisher Scientific). Whole blot images have been included where available as a separate supplemental file.

[0120] Metabolomics To validate the specificity and, thus, the safety of the PT / D-targcting strategy, flux and steady-state metabolite levels were quantified using stable isotope-resolved metabolomics coupled with mass spectrometry.

[0121] Steady-state levels of metabolites: Eyes were collected from 2-month-old experimental and control mice (e.g., before the onset of degeneration), and retinae were harvested and processed as previously described (Zhang L, et al. J Clin Invest. 2016; 126(12):4659-73; Wert KJ, et al. EBioMedicine. 2020;52: 102636).

[0122] Metabolic flux: To confirm the metabolic efficacy of glycolysis following the loss ofPHD, metabolic flux levels were quantified in PT / D-dcficicnt (PHD2,- Pde6yCreERT2 / +;Pdebf"6-"- "6-"- ) and control (P / 7Z>Fi / F L-; Pde6yCreERT2 / +; Pde6fH620Q / H620Q) retinae using stable isotope-resolved metabolomics coupled with gas-chromatography / mass spectrometry (GC-MS). Both in vivo and ex vivo samples were analyzed to reveal how the loss of PHD affected glycolysis, mitochondrial activity, and other potentially relevant metabolic pathways.

[0123] Protocol 1) Quantify rates at which PHD-deficient and control retinae consume glucose medium and release lactate. Retinae were isolated and placed in a Krebs-Ringer Bicarbonate buffer (K.R.B.) supplemented with 5 mM glucose. Every 10 minutes, an aliquot of media was collected and subsequently analyzed for glucose and other metabolite levels with a simple glucose oxidase assay. FIG. 3 shows an example, comparing rates of glucoseconsumption by retinae versus eyecups wherein the neuroretina has been removed (primarily RPE tissue being analyzed).

[0124] Protocol 2) Quantify flux of carbons from universally labeled (U-)13C-glucose into glycolytic and tricarboxylic acid (TCA) cycle intermediates in PHD-deficient vs. control retinae. Retinae were isolated, incubated with 5 mM U-13C-glucose, and harvested at 30 seconds, 90 seconds, 20 minutes, and 40 minutes to analyze glucose consumption and lactate production. Metabolites were extracted with 80% methanol, derivatized for gas chromatography-MS. FIG. 3 shows an example of the quantification of flux into pyruvate, citrate, and lactate. The computational analysis quantified various parameters of isotope enrichment in downstream metabolites including flux of13C from [U-13C] glucose into intermediates of glycolysis and the citric acid cycle and [U-13C] glucose into the pentose phosphate pathway.

[0125] Gene-ablation analysis Human HEK293 and mouse N2A cells were transfected with the plasmid harboring the gRNAs targeting Eglnl or scrambled gRNAs unspecific to any segment of DNA. Genomic DNA from human HEK293 cells and mouse tails was extracted using the DNeasy Blood & Tissue Kit (Qiagen, 69506). DNA was extracted from Human HEK293 and mouse N2A cells after three days of selection, and PCR was performed to validate the gRNA-targeting specificity. gRNA sequences (DNA equivalents) are included in the sequence table provided herein. Plasmid sequences are also included herein.

[0126] Genomic DNA from retinae for subsequent gene-editing analyses was extracted using TRIzol reagent (Thermo Fisher Scientific, 15596018). Genomic PCR was performed using Phusion DNA polymerase (Thermo Fisher Scientific, F549S). Primers for detecting CRISPR- mediated editing in cultured human cells and degenerative mice are provided herein. For deep sequencing, 436- to 267-bp PCR amplicons of sgA+sgB-treated HEK293 cells and AAV -treated mouse retinae were generated using primers with partial Illumina adapter sequences (hEGLN 1 - F-EZ+ hEGLNl-R-EZ for HEK293, and mEGLNl-F-EZ+ mEGLNl-R-EZ for mouse retinae (Sequence Table) and purified using the QIAquick PCR Purification Kit (Qiagen, 28106). Samples were sequenced using an Illumina-based Amplicon-EZ service offered by Genewiz. Between 70,000 and 100,000 next-generation sequencing reads for each sample were generated on 2 x 250 bp paired-end reads. Cas-analyzer software was used to analyze the next-generation sequencing data. The SigmaPlot 14 software package was used to represent the data graphically.All cell lines were validated for chromosome stability and the lack of mycoplasma contamination.

[0127] Therapeutic genome editing of PHD2 in mouse and human cells Using a dual gRNA, exon 1 of Eglnl was ablated in mouse Neuro-2a cells (N2A) and human HEK293 cells. Immunoblots confirmed downregulation ofPHD2 (FIGS. 5D and 5E). Cells transfected with experimental gRNAs directed by CRISPR-Cas9 excision had higher levels of HIF1A expression than cells transfected with control PX459: scramble gRNAs in normoxic conditions. Quantification of mRNA levels was then performed.

[0128] Cone densities Whole eyes were processed for cryosection and subsequent staining, mounting, and imaging. Whole retinas were dissected and flat-mounted. Cone numbers were averaged, assessed, and compared. Regions of the central retina were sampled as this is the region most susceptible to degeneration.

[0129] Adeno-associated viral vectors Two sgRNAs, termed here as sgA and sgB (Sequence Table), together with a GRK1 promoter-driven GFP cassette, were cloned into the pZac2.1 vector (Penn Vector Core PL-C-PV0100, University of Pennsylvania) to form a dual AAV8::U6- gRNAs_F, / / D2; hGRKl GFP construct. For the control vector, the two sgRNA-expressing cassettes of the U6-gRN .\s_PHD2 were replaced with the scrambled sequences, sgC and sgD, which are not homologous to the human or mouse genome. For the AAV-Cas9 construct, a human codon-optimized pyogenes Cas9(SpCas9) was cloned into pZac2.1 between the hGrkl (sCMV) promoter and the synthetic poly(A) sequence. Injection of AAV8::hGRKl-GFP (first virus) allowed the clear marking of ventral subretinal transduction sites. These vectors were then packaged into an AAV8 capsid with a Y733F modification by the Penn Vector Core, University of Pennsylvania. All plasmid sequences are included herein.

[0130] Subretinal injection 2- to 4-week-old mice were anesthetized via i.p. injection with 10 mg / ml ketamine and 1 mg / ml xylazine in PBS at a dosage of 0.1 ml / 10 g B.W. Subretinal injections were performed using a surgical microscope (Zeiss). An incision was made through the cornea from the posterior portion of the right eye using a 31 -gauge needle. A pre-pulled glass micropipette (FIVEphoton Biochemicals, M.G.M. ID) connected to a 1 ml syringe (B.D., 309623) by the tubing of a butterfly infusion set was inserted through the corneal incision and pushed through the choroid and retinal pigment epithelial cell layer. Each eye received 1.5 pl ofdual AAV mixture (5 x 1012 vg / ml of each virus) or PBS. In each group, 1 / 10 volume of AAV8- CMV::H2B-EGFP (5 x 1012 vg / ml) was co-injected to ensure successful delivery.

[0131] Statistics All statistical tests utilized a standardized two-sample t-test with equal variance assumptions, where appropriate. An exception is when a contralateral eye is a control, wherein a paired t-test was implemented instead. All error bars are S.E.M. unless explicitly denoted otherwise. All ERG results are averaged, with each mouse representing n = 1. The exception is in the case of the contralateral eye serving as a control, wherein the paired t-test is implemented, and each eye is taken as n = 1. All RNA and Protein analysis considers a single retinal extract as n = 1. All data were analyzed in PRISM GraphPad comprehensive statistical analysis software.Example 1Pharmacological inhibition of PHD elevates aerobic glycolysis and prolongs photoreceptor survival.

[0132] Doses of the PHD inhibitor, FG-4592, were administered by oral gavage to a mouse model for RP that has a PDE6 deficiency (Pde6 / iH620^ / H620') and degenerates on a timeline similar to the Pde6prdI0model. Doses were delivered every other day from P5, boosting aerobic glycolysis in rods and cones. Following administration of the systemic PHD inhibitor, steadystate levels of some glycolytic intermediates in retinae harvested from FG-4592 treated mice were significantly higher than in retinae from control mice (FIG. 1A). Tricarboxylic acid (TCA) cycle intermediates were not significantly different between experimental and control groups (FIG. 1A). Histological analysis at age P35 of treated and control eyes showed an increased thickness of the outer nuclear layer (ONL) in the Pde6p,l62ll~,l62ll~ RP model treated with 20 mg / kg FG-4592 compared to control untreated mice (FIGS. IB and 1C). At four weeks of age, phototransduction was evaluated by electroretinography (ERG). Photopic and mixed b-wave responses were significantly enhanced in treated mice compared with untreated controls (FIGS. ID and IE). Rod b-wave responses remained unchanged.Example 2Preclinical model development and tamoxifen-induced ablation of PHD 1,2, 3

[0133] The effects of PHD inhibition were evaluated on photoreceptor resilience by eliminating expression in the retina of all three PHD isoenzymes. The ability of this cell-specific ablation to rescue degeneration was probed in Pde^p'1620- '1620Pde6yCreERT2l+mice, a well-established preclinical model of arRP. Pde / ip"621'- "621'- ', Pde6yCreERT2'+mice carry a rod photoreceptor-specific tamoxifen-inducible Cre driver [Pde6yCreERT2 / +}. Pde6fiH620Q / H620Q,- Pde6yCreERT2l+animals were intercrossed with the conditional PHD12, 2, 3? mouse line to generate the experimental mouse line. Tamoxifen treatment blocks expression of PHD2, 1, and 3 (Eglnl, 2, and 3), which encode all of the PPID isoenzymes. PHD2Pde6yCreERT2 / +,' Pde6[),l62lppH62l’d mice served as the experimental group and were given an i.p. injection of tamoxifen for three consecutive days (P9, PIO, and Pl 1) following the completion of retinal cell differentiation. Control PHD'12]-. Pde6yCreERT2 / +; Pde6 / 3H620Q / H620Qmice received a sham injection of 10% Ethanol (w / w) in sunflower oil at the same age as treated groups.

[0134] To demonstrate that PHD1, 2, and 3 were ablated following tamoxifen treatment, genomic DNA was isolated from mouse retinae and the PHD genes (Eglnl, 2, 3) were analyzed with PCR. Genomic PCR revealed a 481 -bp truncated band in addition to the 900-bp parental fragments in the treated retinae (FIG. 8A). The 900-bp parental product is less intense because, in addition to rods, the whole retina contains cones, bipolar cells, et al., in which the Pde6yCreERT2driver was inactive. Primers for genomic PCR are included herein. Control retinae in the treated group presented only the non-truncated wild-type fragment (900bp). Genomic PCR blots are shown to validate PHD2 and 3 deletions (FIGS. 8B and 8C).Example 3 Upregulation of aerobic glycolysis in PHDl,2,3-delicient retinae

[0135] Because of the critical role of PHD1, 2, and 3 in regulating aerobic glycolysis, extracts from retinae deficient in PHD1, 2, and 3 were collected, and the transcripts of downstream glycolytic genes regulated by the P / 7D-VHL-HIF aerobic axis were quantified via qPCR. mRNA expression was quantified to assess changes between PHD1,2,3~2~; Pde6ftn620~n62r'~ and PHD 1,2, 3loxP / loxP;Pde6pn2E2(-m<EJ'()at three weeks of age, prior to the onset of ONL loss.Quantification of mRNA transcript levels from samples collected from the retinae of experimental and control mice revealed that Glutl, Glut3, Pfk, Hk2, and Pdkl increased in the PHD 1, 2,3^-; Pde6pH620Q / H620Qmice compared with controls (FIG. 2A). PHDl,2,3loxP / loxP; Pde6ftn620~H62r'~ degenerate age-matched mice served as a control. p-Actin served as a loading control. Immunoblots were treated with antibodies against downstream targets to PHD1, PHD2, and PHD3, and subsequent analysis of glycolytic regulators in experimental and control retinal extracts revealed higher protein levels of hypoxia-inducible factors (HIF2A), glucosetransporters (GLUT1), and hexokinase (HK2) in experimental mice (FIG. 2B). Increased aerobic glycolytic shifts were consistent with the expected result of PHD suppression. This data is quantified, and magnitudes of protein expression are compared (FIG. 2C). Efficient, nearcomplete rod-specific Pde6yCreERT2-mediated downregulation of PHD expression via HIF levels and subsequent aerobic glycolytic shifts were observed (FIG. 2C). PHD levels were difficult to assay from tissue, likely due to their rapid degradation in vivo, however genomic deletion was confirmed previously.Example 4Analysis of downstream glycolytic metabolites following PHD1,2,3 ablation

[0136] The qPCR data, and immunoblots from tamoxifen-treated versus untreated Pde6l>'1620- '1620~ mice suggested that loss of PHD expression could influence glycolytic flux. This metabolic flux was measured through glycolysis and the TCA cycle to clarify the influence of PHD on retinal metabolism. Retinae were isolated and incubated with U-13C glucose for 30 or 90 seconds. Metabolites were extracted and derivatized, and gas chromatography / mass spectrometry was used to quantify incorporation of carbons from13C glucose into glycolytic and TCA cycle intermediates (FIG. 3). More rapid labeling and higher accumulation of glycolytic intermediates was found all the way through pyruvate in PF / D-deficient Pded / i11620^620^ retinae (dashed lines) compared to control Pde6l>'1620- '1620~ retinae (solid lines). The most substantial effect is diminished flux into and through the TCA cycle, suggesting that elevated PDH kinase (PDK1) expression (FIGS. 2A and 9) may inhibit oxidation of pyruvate to acetyl CoA. Surprisingly, the overall rates of glucose depletion from the media and lactate release into the media are unaffected. Pentose Phosphate Pathway activity was probed and did not indicate an increase in FT / D-dcficicnt retinas (FIG. 10). This data directly supports the accumulation of M3 labeled Pyruvate and corresponding depletion of M2 Citrate seen in FIG. 3. Overall levels of recoverin were probed as a proxy for rod survival in metabolic tracing experiments, and indicated that the differences observed were not due to overall rod preservation differences (FIG. 11).Example 5 Tamoxifen-induced ablation of PHD2 enhances photoreceptor survival

[0137] After validating tamoxifen-induced excision of PHD1, 2, 3 and confirming that parts of glycolytic flux are enhanced, neurological function was analyzed by electroretinography. Atthree weeks post-treatment, experimental mice responded with significantly larger b-wave amplitudes than control mice (FIGS. 4A-4C). Representative traces are shown in FIG. 12A Histological analysis revealed that the outer nuclear layer (ONL) in experimental mice is significantly thicker than in control mice at 500 pm from the optic nerve head (FIG. 4D). Comprehensive spider plots indicated that the rescue effect was strongest near the optic nerve head, but was preserved throughout almost the entirety of the retina at both four and six weeks of age (FIGS. 4E and 4F). Fluorescent flat-mount staining of cone photoreceptors in the retinae of experimental and control mice revealed more significant numbers and a greater density of cone cells labeled with anti-peanut agglutinin antibodies in experimental mice retina than in controls (FIGS. 4G and 4H). No safety concerns such as neovascularization were detected in any of the Pde6l>'1620- '1620~ retinae lacking PHD1, 2, 3 up to a time of 1 year of age. Fluorescein angiography (FA) images from 1 year old, subretinally -transduced (at an age of 1 -month) RHOCIIOR / +mice demonstrated no patterns of neovascularization and support a lack of safety concerns (FIG. 13).Example 6 Therapeutic genome editing of PHD2 in mouse and human cells

[0138] Using a dual-gRNA guided CRISPR Cas9 system, exon 1 of PHD2 was ablated in mouse Neuro-2a cells (N2A) and human HEK293 cells (FIG. 5A). The overwhelming majority of resultant quantified edits were indels that were not divisible by three, thereby causing a frameshift mediated premature stop codon, ultimately truncating the gene expression (FIGS. 5B and 5C). Unsuccessful edits are indicated with an Asterix (*). Cells transfected with experimental gRNAs directed by CRISPR-Cas9 excision had markedly lower PHD2 protein expression than cells transfected with control PX459: scramble gRNAs (FIGS. 5D and 5E). Results indicated successful PHD2 ablation and associated increased HIF levels as anticipated, compared to a non-transfected WT control (FIG. 5E). Bands were quantified in ImageJ and compared and showed a ~1.7 fold increase in the treated cells compared to non-edited control cells. Subsequent quantification of mRNA levels revealed that the cells transfected with experimental gRNAs also had higher levels of LDHA, PDK1, GLUT1, and GLUT 3, indicating that PHD2 ablation may contribute to an increase in glycolysis (FIG. 5F).Example 7Therapeutic genome editing of PHD2 in arRP and adRP preclinical models31

[0139] Following the confirmation of the therapeutic PHD2 editing in human cells, wild-type C57BL / 6J mice were transduced subretinally with dual gRNA AAV-vectors targeting rod photoreceptors to validate the transduction efficiency of AAV8 delivery. Whole retinae were dissociated and FACS was used to identify AAV-transduced rod photoreceptors, utilizing CD73+ markers as a proxy for this cell type. Approximately 35% of cells positive for CD73+, a rod-specific cell surface marker, also were positive for GFP, indicating that they had been transduced by AAV8:: / zG7?A7-GFP (FIGS. 6A-6C). Few other CD73 -negative, GFP -positive cells were present, confirming that this AAV8 had driven expression specifically in rod photoreceptors. GFP+ / CD73+ cells were absent in control-injected mice. The specificity of this AAV8 vector has been previously confirmed.

[0140] After confirming the specificity and efficiency of the AAV8 in transducing rod photoreceptors, the next step in developing this therapeutic was to evaluate efficacy in the Pde^pH620Q / lpde6PCas9arRP model. This model harbors Cas9 expressed in photoreceptors and degenerates on a timeline akin to homozygous Pdebp'1620 1620~ mice. Importantly, Pde6fiCas9 / +photoreceptors have no detectable deleterious effects on their own (FIG. 14).Electroretinography (ERG) at eight weeks of age was used to quantify photo-responses from Pdebp1162'1-2116211- mice transduced with the therapeutic PPID2 editing vector, AAV8::U6- gRNAs_P / / D2 at 2 weeks of age. Co-injection of AAV8:: / zG7?A7-GFP revealed that the subretinal transduction area covered -25% of the retina. Contralateral eyes served as noninjected controls (FIG. 7A). ERG tracings showed that the therapeutic PHD2 editing vector enhanced photoreceptor function (FIG. 7B). These traces were analyzed for maximal rod and cone responses, a metric routinely used as a hallmark of retinal function, which revealed that magnitudes of a- and b-waves from the injected eyes generated significantly larger ERG responses than the control noninjected contralateral eyes (FIG. 7C). Eight-week-old wild type responses can be seen in FIG. 12B, which helps better appreciate the discrepancy between the treated and control groups. The histology preservation effect remains to be further characterized in this mouse model.

[0141] Following the increased functional preservation from therapeutic PHD2 editing in an arRP preclinical model, the AAV therapeutic PHD2 editing vector was applied to an alternative degeneration model. An approach identical to the one used for the arRP model (Pdebp- deficiency) was used as a treatment for an adRP model caused by a rhodopsin mutation,Rhocn0RJ+. Subretinal injections of AAVS^Ub-gRNAs P / YDZ were performed into 4-week-old Rhocn0R / +mice and observed a similar ventral area of AAV transduction (FIG. 7D), readily identifiable by co-inj ection with a GFP-cassette containing AAV, similarly to the approach in the previous model. ERG analyses showed that photo-response amplitudes from rods in treated eyes were significantly improved compared to noninjected control eyes at six months (representative recordings are shown in FIG. 7E). ERG responses were quantified, averaged, and compared, revealing a statistically significant rescue of function (FIG. 7F). Lastly, histology showed increased structural preservation in the AAV8-transduced peripheral retina compared to the noninjected contralateral retina, suggesting that the rescue effect extended to retinal function and survival (FIGS. 7G and 7H). Interestingly, the preservation effect seemed to spread to the central retina (500pm from optic nerve head) as seen in FIG. 7G, even though the area of GFP seen in FIG. 7D is further away from the central retinal region. This is likely accountable to a fluctuation in the area of viral transduction, and some degree of variability in this platform. These findings demonstrate that 7V / D2-ablation can rescue degeneration caused by two distinct types of genetic deficiency, an essential factor for a truly gene-agnostic therapy.Sequences

[0142] The scope of the present invention is not limited by what has been specifically shown and described hereinabove. Those skilled in the art will recognize that there are suitable alternatives to the depicted examples of materials, configurations, constructions, and dimensions. Variations, modifications, and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and scope of the invention.

[0143] Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety.

Claims

CLAIMSWhat is claimed is:

1. A method for increasing retinal cell survival and / or preserving retinal function comprising contacting a retinal cell with a prolyl hydroxylase domain-containing protein 2 (PHD2) gene editing system, or one or more nucleic acids encoding thereof, wherein the PHD2 gene editing system comprises a CRISPR / Cas gene editing system, and wherein the CRISPR / Cas gene editing system comprises two gRNA of SEQ ID NOs: 11 and 12.

2. The method of any of claim 1, wherein the two gRNAs are provided in a crRNA array.

3. The method of any of claims 1-2, wherein the retinal cell is a rod cell and / or a cone cell.

4. The method of any of claims 1-3, wherein the cell is in a subject.

5. The method of claim 4, wherein the method comprises administering the PHD2 gene editing system, or one or more nucleic acids encoding thereof, to the subject.

6. The method of claim 5, wherein administration is by intravitreal or subretinal injection.

7. The method of any of claims 4-6, wherein the subject has or is suspected of having one or more retinal degenerative diseases.

8. The method of claim 7, wherein the one or more retinal degenerative diseases comprise retinitis pigmentosa (RP), age-related macular degeneration (AMD), glaucoma, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Lewy body dementia, or combinations thereof.

9. The method of any of claims 1-8, wherein the one or more nucleic acids comprise a recombinant adeno-associated viral (AAV) vector.

10. A gene editing system for ablating a prolyl hydroxylase domain-containing protein 2 (PHD2) gene, comprising: an RNA guided nuclease; and two guide RNAs (gRNAs) of SEQ ID NOs: 11 and 12 configured to target exon 1 of the PHD2 gene, orone or more nucleic acids encoding the RNA guided nuclease and the two gRNAs of SEQ ID NOs: 11 and 12.

11. The gene editing system of claim 10, wherein the two gRNAs are provided in a crRNA array.

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