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Definitions

• Retinal degenerative diseases are characterized by the degeneration of one or two retinal cell types, such as retinal pigment epithelial (RPE) and/or photoreceptor cell loss in age-related macular disease (AMD) and retinitis pigmentosa (RP), and retinal ganglion cell (RGC) death in glaucoma.
• RPE retinal pigment epithelial
• AMD age-related macular disease
• RP retinitis pigmentosa
• RRC retinal ganglion cell
• RPE cells Aberrant RPE cells can be observed through optical coherence tomography (OCT) in AMD patients.
• OCT optical coherence tomography
• RP retinitis pigmentosa
• Stargardt's disease or juvenile macular degeneration which is characterized by the loss of the RPE and photoreceptors in the macular area, causes central vision loss at an early age.
• Loss of retinal ganglion cells (RGCs) can be observed in patients with glaucoma. Once the retinal cell degeneration is triggered, no treatments can reverse it.
• rods and cones are responsible for dim light vision and daylight vision (including color), respectively.
• Rods are located mainly in the peripheral retina, and cones are concentrated in a small portion of the retina, the macula, which provides high-resolution central vision.
• Bipolar cells receive visual signals from photoreceptor cells and transmit these signals to RGCs coordinated by horizontal and amacrine cells. Ultimately, all the signals are gathered in the optic nerve and transmitted to the brain. Both cell degeneration and synaptic disruption can cause permanent visual impairment or even blindness. See, Jin 2019.
• AMD Age-related macular disease
• AMD is the leading cause of vision loss in Europe, the United States, and Australia. Almost two-thirds of the population over 80 years of age will have signs of AMD resulting from the wet or exudative form, which is characterized by the presence of drusen and CNV (subfoveal choroidal neovascularization).
• AMD is a chronic progressive disease characterized by damage to the central retina zone. Changes in choriocapillaries, retinal pigment epithelium (RPE), and Bruch’s membrane (typical for aging) underlie AMD pathogenesis; however, the mechanisms launching the transfer of typical age-related changes in the pathological process are unknown.
• “Dry” and “wet” forms of the disease are categorized. Approximately 90% of cases are of the “dry” atrophic form of AMD; today, there is no method for its treatment. In the “dry” form of AMD, drusen are diagnosed in the macular area, pigment redistribution occurs, defects of pigment epithelium and the choriocapillary layer appear, and death of photoreceptors occurs against a background of RPE cell atrophy. “Wet” (exudative) form develops in -10% of AMD patients and is characterized by ingrowing of newly generated vessels through Bruch’s membrane defects under the retinal pigment epithelium or under neuroepithelium.
• the model has proven to be suitable for testing the efficacy of new drugs through systemic or local (intraocular) administration and has shown predictive value for drug effects in patients with AMD, for example, with vascular endothelial growth factor receptor (VEGFR) trap or anecortave acetate (Telegina 2017).
• AMD vascular endothelial growth factor receptor
• VEGF vascular endothelial growth factor
• bevacizumab (Genentech Inc., South San Francisco, CA; commercialized worldwide by Roche) is a full-length humanized antibody that binds and blocks all VEGF isoforms.
• Oxidative stress can trigger apoptosis, which may activate and recruit macrophages and induce inflammation.
• Apoptosis may be one of the triggers of choroidal inflammation and consequently angiogenesis in CNV model (Du 2013).
• X-linked RP The most common form of recessive RP is X-linked RP, which is caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. Talib M et al. (2016). X-linked RP is characterized by the degeneration of rod and cone photoreceptors during childhood, leading to visual field constriction and severe sight loss at an early age. Despite promising results in animal models (Bennett J (2017); Boye SE et al. (2013); Sahel JA et al. (2014); Vandenberghe LH (2015)), gene therapy targeting photoreceptor degeneration in humans for RPGR related RP has not been fully developed until now.
• RPGR retinitis pigmentosa GTPase regulator
• NDD Neurodegenerative disorders
• central nervous system conditions including Parkinson’s disease and Alzheimer’s disease can affect patients’ vision.
• Parkinson’s disease and Alzheimer’s disease can cause direct or retrograde degenerations of the optic nerve, retinal cells, and surrounding visual structures. While not often obvious, these conditions affect coordination, mobility, and visual perception, resulting in increased risks of falls and related injuries.
• AIMP2-DX2 is an alternative, antagonistic splicing variant of AIMP2, which is a multifactorial apoptotic gene.
• AIMP2-DX2 is known to suppress cell apoptosis by hindering the functions of AIMP2.
• AIMP2-DX2, acting as competitive inhibitor of AIMP2 suppresses TNF- alpha mediated apoptosis through inhibition of ubiquitination/degradation of TRAF2.
• AIMP2-DX2 has been confirmed as an existing lung cancer induction factor and, in the existing research, it was confirmed that AIMP2-DX2, manifested extensively in cancer cells, induces cancer by interfering with the cancer suppression functions of AIMP2.
• manifestation of AIMP2-DX2 in normal cell progresses cancerization of cells while suppression of manifestation of AIMP2-DX2, suppresses cancer growth, thereby displaying treatment effects.
• AIMP2-DX2 can be useful in treating neuronal diseases (KR10-2015-0140723 (2017) and US2019/0298858 (2019). SUMMARY OF THE INVENTION
• nucleic acid molecule comprising an exon 2-deleted AIMP2 variant (AIMP2-DX2 or DX2).
• the nucleic acid molecule is a viral vector or a nonviral vector, e.g., a recombinant vector.
• a recombinant vector comprising an exon 2-deleted AIMP2 variant (AIMP2-DX2 or DX2) gene.
• the nucleic acid molecule is administered by a chemical conjugation with Galnac, a cell-penetrating peptide, a nucleic acid or glycopeptide mediated tool, a synthetic compound vector; a lipid-mediated delivery such as lipid nanoparticle encapsulation; an inorganic vector mediated delivery such as inorganic vectors; a biological delivery such as exosome mediated delivery; or a physical delivery such as by micro/nano needles, pressure -perfusion, microprojectiles, electrical energy/electroporation/iontophoresis, sonoporation, magnetoporation, or optoporation/photodynamic energy.
• the retinal degenerative disease is retinitis pigmentosa, Leber’s congenital amaurosis, Cone -rod dystrophy, glaucoma, or diabetic retinopathy.
• the retinal degenerative disease precedes or is accompanied by Parkinson’s disease, Alzheimers’s disease, or amyotrophic lateral sclerosis.
• the retinal degenerative disease is not age-related macular disease.
• the methods of treating retinal degenerative diseases does not include treating age-related macular disease (AMD).
• AMD age-related macular disease
• the AMD is wet AMD.
• the AMD is dry AMD.
• the recombinant vector can further comprise an miR-142 target sequence.
• the vector can further comprise a promoter operably linked to the AIMP2-DX2.
• the promoter is a Retrovirus (LTR) promoter, cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, MT promoter, EF-1 alpha promoter, UB6 promoter, chicken beta-actin promoter, CAG promoter, RPE65 promoter or opsin promoter.
• the miR-142 target sequence can be 3’ to the AIMP2-DX2 gene.
• the AIMP2-DX2 gene comprises a nucleotide sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:2, 13, 14, 15, 16, 17, 18, 19, or 20.
• the AIMP2-DX2 gene comprises a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:2, 13, 14, 15, 16, 17, 18, 19, or 20.
• the AIMP2-DX2 gene does not have an exon comprising a nucleotide sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NOTO or 11.
• the AIMP2-DX2 gene does not have an exon comprising a nucleotide sequence encoding an amino acid sequence of SEQ ID NOTO or 11.
• the miR-142 target sequence can comprise a nucleotide sequence comprising AC ACTA.
• the miR-142 target sequence comprises ACACTA and 1-17 additional contiguous nucleotides of SEQ ID NO:5.
• the miR-142 target sequence comprises a nucleotide sequence at least 50% identical to a nucleotide sequence of SEQ ID NO:5 (TCCATAAAGTAGGAAACACTACA; miR-142-3p target sequence).
• the miR-142 target sequence can comprise a nucleotide sequence of SEQ ID NO:5.
• the miR-142 target sequence comprises ACTTTA.
• the miR-142 target sequence comprises ACTTTA and 1-15 additional contiguous nucleotides of SEQ ID NO:7. In some embodiments, the miR-142 target sequence comprises a nucleotide sequence at least 50% identical to a nucleotide sequence of SEQ ID NO:7 (AGTAGTGCTTTCTACTTTATG; miR-142-5p target sequence). In some embodiments, the miR-142 target sequence comprises a nucleotide sequence of SEQ ID NO:7.
• the miR-142 target sequence can be repeated 2-10 times in the vector disclosed herein.
• the vector can be a viral vector.
• the viral vector can be an adenovirus, adeno-associated virus, lentivirus, retrovirus, human immunodeficiency virus (HIV), murine leukemia virus (MLV), avian sarcoma/leukosis (ASLV), spleen necrosis virus (SNV), Rous sarcoma virus (RSV), mouse mammary tumor virus (MMTV), Herpes simplex virus, or vaccinia virus vector.
• the recombinant vector is administered topically to, by intravitreal injection to, by subconjunctival injection to, or into a subretinal space of the subject.
• the methods disclosed herein can further comprise administering to the subject an additional therapeutic agent.
• the additional therapeutic agent is ranibizumab, aflibercept, or bevacizumab.
• FIG. 1 An example recombinant vector.
• FIG. 2 Nerve cell-specific expression of a recombinant vector under an in vitro environment.
• FIG. 3 An miR142-3pT (target) sequence with 4 repeats of miR142-3pT (underlined) (SEQ ID NO:6).
• FIG. 4A A schematic of miR142-3pT with lx, 2x, and 3x repeats, and mutant.
• FIG. 4B shows miR142-3p inhibition on DX2 expression with lx, 2x, and 3x repeats of miR-142-3pT.
• FIG. 5 A core binding sequence is important in DX2 inhibition.
• a vector with Tseq x3 repeats, which showed significant inhibition of DX2 (FIG. 4B), and DX2 construct were used as controls.
• 100 pmol of miR-142-3p treatment inhibited Tseq x3 vector significantly but DX2 and mutant sequence were not inhibited.
• FIGS. 6A-6C A comparison of the amino acid sequences of AIMP2-DX2 and variants (FIGS. 6B and 6C are continuations of FIG. 6A).
• FIGS. 7A-7I DX2 physically binds with AIMP2 to decrease PARP-1 activation.
• FIG. 7A AIMP2, and DX2 expression was induced by transfection of each plasmid in SH-SY5Y cells and then followed by analyses of PARP-1 pull-down assays. DX2 shows a higher affinity for PARP-1 than AIMP2.
• FIG. 7B SH-SY5Y cells were transfected with vehicle (Con) or DX2 expression (DX2) plasmid, incubated for 24 hours and lysed.
• FIG. 7C DX2 siRNA (DX2) and control siRNA (Con) were transfected into SH-SY5Y and incubated for 48 hours. Total cell lysates were incubated with protein agarose beads to immunoprecipitate PARP-1 bound AIMP2 and analyzed by immunoblot analysis.
• FIGS. 7D and 7E GFP- or Myc-tagged AIMP2 and/or DX2 expressing plasmid was transfected into HEK 293 cells and the binding affinity was measured by immunoprecipitation (IP) with myc antibody (FIG. 7D) and GFP antibody (FIG.
• FIGS. 7F and 7G SH-SY5Y cells were transfected with the EV (empty vector), AIMP2 and DX2 expression plasmid, and 24 hours later, the transfected cells were incubated with or without 10 400 pM H2O2 for 4 hours. Cleaved PARP-1 levels (FIG. 7F) and PARlyation (FIG. 7G) were examined using immunoblot assays. In oxidative stressed-induce cellular damage conditions, DX2 attenuates cleavage of PARP-1 (FIG. 7F) and PARylation (FIG. 7G) related to cell death.
• FIG. 7H SH-SY5Y cells were transfected with the EV (empty vector), AIMP2 and DX2 expression plasmid, and 24 hours later, the transfected cells were incubated with or without 10 400 pM H2O2 for 4 hours. Cleaved PARP-1 levels (FIG. 7F) and PARlyation (FIG. 7G
• FIG. 71 Schematic figure of AIMP2-induced PARP-1 activation. In the absence of DX2, AIMP2 dimer induces PARP-1 activation and neuronal death. However, in the presence of DX2, DX2 interacts with AIMP2 and inhibits PARP- 1 activation.
• FIGS. 8A-8C DX2 reduces neurotoxin-induced neuronal cell death.
• FIG. 8A N2A cells were transfected with EV, AIMP2 and DX2 expression plasmids and the transfected cells were incubated with or without H2O2 for 4 hours, then the cell viability was measured using MTT.
• FIGS. 8B and 8C SH-SY5Y cells were co-transfected with EV, AIMP2 and DX2 expression plasmids, incubated with H2O2 for 4 hours and the cell viability was measured by MTT analysis. The number shows the amounts of transfected DNA.
• FIGS. 9A-9C DX2 compromises pro-apoptotic activity of AIMP2 via the competitive inhibition of AIMP2 binding to TRAF2.
• FIG. 9A HEK293 cells were transfected with the indicated amount of Myc- DX2 and -AIMP2. The expression of endo- and exo-AIMP2 and -DX2 was confirmed by western blotting of whole cell lysates (WCL) with anti-AIMP2 antibody (lower panel). TRAF2 was immunoprecipitated with its specific antibody and AIMP2 or DX2 bound to TRAF2 were detected with anti-Myc antibody (upper panel). Choi JW et al. (2012).
• FIG. 9B DX2 increased the NF-kB activity, whereas AIMP2 gave the opposite effect.
• FIG. 9C DX2 overexpression abolished the TNF-a induced cell death.
• FIGS. 10A-10B Overexpression of DX2 reduces TNFa-induced neuronal cell death.
• FIG. HA N2A cells were transfected with EV and DX2 and treated with TNF-a and CHX for 6 h. Cell viability was evaluated by the MTT assay. # P ⁇ 0.05.
• FIG. 10B Primary neuronal cells in WT mice were transfected with EV (empty vector) and DX2, and the MTT assay was performed to assess their viability. # P ⁇ 0.0.
• FIGS. 11A-11B AAV2-GFP transduction efficacy test.
• FIG. HA Transduction efficacy test of AAV-GFP.
• the SK-SY5Y cells were infected with scAAV-GFP or ssAAV-GFP, and 48 hours later, (FIG. 11B) GFP expression was observed on fluorescence microscopy.
• FIGS. 12A-12G AAV2-DX2 has anti-apoptotic effects and alters cellular signaling.
• FIG. 12A Cytotoxic effects in the primary neurons (Neuron), MEF (Mouse embryonic fibroblasts), hepatocytes, and MSC (Mesenchymal stem cells). After H2O2 treatment, a decreased cytotoxic effect was observed in DX2 transduced cells (AAV2-DX2) when compared with control- transduced cells (c).
• FIG. 12B DX2 is not required for normal cell growth in SH-SY5Y (left) and primary neuronal cells (right).
• FIG. 12C Enrichment plot of RNAseq of AAV-GFP or AAV2-DX2-infected neuroblastoma cells SK-N-SH and N2A.
• FIGS. 12D and 12E Graph of gene counts representing cell death and inflammatory related pathways were downregulated.
• FIGS. 12F and 12G p-value plot of the signaling pathway changed by DX2 overexpression, ns non-significant; *P ⁇ 0.05; **, P ⁇ 0.01; ****, P ⁇ 0.0001, t-test.
• FIG. 13 DX2 increased retinal photoreceptor neuronal cell viability. 661W cells were seeded at 5 x 10 A 4 cells per well in a 96-well plate, followed by infection with AAV-DX2 virus at an 5000MGI. After 48 hours, cells were treated with varying concentrations of H2O2. Cell viability was assessed 24 hours later using the MTT assay. [0050] FIG. 14. DX2 reduces the levels of BAX induced by H2O2. 661W cells were infected with either AAV-GFP or AAV-DX2 at an MOI of 5,000 each. After 72 hours, cells were treated with 2 mM H2O2 for 6 hours, and then the RNA levels of BAX were assessed.
• FIGS. 15A-15B Overexpression of DX2 results in an increase in 67LR levels in 661W cells.
• FIG. 15 A Empty vector (EV) and Strep-DX2 (DX2) were transfected into 661W cells using Lipofectamine 3000 reagent. DX2 transfection resulted in an increase in 67LR levels.
• FIG. 15B Immunofluorescence staining was performed on 661W cells under the same conditions as in FIG. 15 A.
• FIG. 16 DX2 binds to KRS similarly to AIMP2.
• Cells transfected with 2 pg of Flag-tag- AIMP2 and Flag-tag-DX2 were subjected to cell lysis using NP-40 lysis buffer. Subsequently, a mixture of 15 pl agarose A/G plus beads and KRS antibody was used for the pull-down assay.
• FIGS. 17A-17B DX2 modulates the levels of KRS membrane form under ROS conditions, leading to an increase in 67LR levels.
• FIG. 17A 661W cells were infected with AAV-GFP and AAV-DX2 at a multiplicity of infection (MOI) of 5,000, followed by treatment with 2 mM H2O2 for 6 hours after 72 hours post-infection. Subsequently, cell lysis was performed and changes in KRS and 67LR levels were observed using Western blot analysis.
• FIG. 17B Following the same experimental conditions as in FIG. 17 A, cells were fractionated into cytosolic and membrane components, and Western blot analysis was conducted. Upon H2O2 treatment, a reduction in the membrane form of KRS was observed, while treatment with AAV-DX2 resulted in an increase in the membrane forms of both 67LR and KRS.
• FIG. 18A Cross-sectional histology (H&E staining) of retina.
• FIG. 18B Retina thickness.
• FIG. 18C RPE (Retinal Pigment Epithelial) thickness.
• FIG. 18D ONL (Outer Nuclear Layer of Photoreceptors) thickness.
• FIG. 18E Outer Segment thickness.
• FIG. 18F OPL Outer Plexiform Layer) thickness. All the samples were acquired from the optic nerve containing section with 10 pm thickness.
• FIG. 18G Integrity and proliferation of RPE (Retinal Pigment Epithelial).
• Fig. 18H PR (Photoreceptor) recovery.
• FIG. 18J-18O Functional recovery of retina.
• Electroretinograph of normal ERG graph format (FIG. 18J-18K).
• A-wave amplitude (FIG. 18L) and A-wave latency (FIG. 18M)
• b-wave amplitude (FIG. 18N)
• b-wave latency (FIG. 180).
• FIG. 19 A Fluorescein angiography and indocyanine green angiography images of laser photocoagulation sites after treatment with scAAV2-GFP or scAAV2-DX2.
• FIG. 19E is
• VEGF laser-induced choroidal neovascularization
• FIGS. 20A-20B Retinal pigment epithelium (RPE) degenerated area Fundus photography.
• FIGS. 20C-20F The DX2 expression level of test articles (AAV2-DX2) treated groups.
• FIGS. 20G-20J Immunohistochemistry.
• FIGS. 20K-20L Electroretinogram.
• FIGS. 21A-21B Electroretinogram measurements of A wave (FIG. 21 A) and B wave (FIG 21B).
• FIGS. 22A-22B AAV2-DX2AAV2-DX2 prevents neovascularization in Laser-induced choroidal neovascularization (CNV) rabbit model.
• FIG. 22A The fluorescein intensity.
• FIG. 22B Isolectin B4 staining.
• FIGS. 23A-23M Biodistribution study to assess DX2 expression in whole-body organs.
• FIGS. 23 A qPCR analysis to confirm expression of DX2 in injected tissue area.
• FIGS. 23B-23M Analysis of DX2 expression in thymus, liver, testis, brain, spleen, kidney, heart, stomach, mesenteric lymph node, spinal cord, pancreas, and lung using qPCR.
• NDD Neurodegenerative disorders
• Inherited retinal disease is a collective concept that refers to heterogeneous eye diseases that are caused by dozens of mutations in various genes and vary in presence and frequency depending on race and region.
• gene therapy to replace a mutated gene with normal WT gene has emerged as a promising treatment method.
• most of the gene therapy drugs that are currently undergoing clinical phase studies are therapy through the introduction of a single normal WT gene to replace the mutant gene, which is a limit that can only be expected to be effective in a very limited number of patients.
• the clinical study outcome of single mutant gene replacement gene therapy e.g., Luxturna
• Parkinson’s disease is a progressive neuronal degenerative disorder. Destruction of dopaminergic neurons in the substantia niagra with a consequent reduction of dopamine actions in the corpus striatum, parts of the basal ganglia system that are involved in motor control is thought to be a pathophysiologic mechanism of Parkinson’s disease. While Parkinson’s disease is primarily characterized by tremors, rigidity, and postural instability, vague visual symptoms such as blurred and double vision, uncontrolled eye movements, light sensitivity, eye strain, and difficulty reading are common. Mild ocular motor abnormalities occur in as many as 75 percent of patients with idiopathic Parkinson’s disease but often are left untreated. Yet because these symptoms can further reduce a patient’s already jeopardized quality of life and functionality, they warrant specialized treatment.
• ALS Amyotrophic lateral sclerosis
• ophthalmic complaints are not presently considered a classic symptom of ALS
• retinal changes such as thinning, axonal degeneration and inclusion bodies have been found in many patients.
• Retinal abnormalities observed in postmortem human tissues and animal models are similar to spinal cord changes in ALS.
• These findings are not dramatically unexpected because retina shares an ontogenetic relationship with the brain, and many genes are associated both with neurodegeneration and retinal diseases. See Soldatov 2021.
• Alzheimer’s disease is the most common form of dementia, and it is characterized by destroyed and damaged connections between neural cells.
• P-amyloid protein in the form of amyloid plaques and/or neurofibrillary tangles composed of aggregated tau protein.
• Ocular degeneration in Alzheimer’s disease occurs as the retinal nerve fiber layer thins. Neurons responsible for visual processing tend to be damaged more than primary vision neurons, resulting in ambiguous vision symptoms in the early stages of the disease. Deficits in recognizing objects, seeing colors, and processing visual motion can occur at any stage, increasing the risk of accidents such as falls, lacerations, and burns.
• DX2 an exon 2-deficient antagonistic splicing variant of AIMP2, effectively compromises AIMP2-induced neuronal death.
• AIMP2 is one of three auxiliary proteins that form the multi- tRNA synthetase complex. Besides their role in supporting tRNA ligation during translation, AIMP2 has been reported to play as a multi-functional and potent cell death-inducing gene.
• AIMP2 over-activates cell apoptotic signaling pathways such as PARP-1 or p53 or suppresses cell survival signaling pathways such as TRAF signaling or LR expression.
• DX2 acts as an antagonistic competitor against AIMP2, consequently directly and indirectly inhibits AIMP2 induced cell death.
• DX2 inhibits PARP-1 and p53-induced neuronal death in Parkinson’s disease model.
• Overexpression of DX2 using AAV in the substantia nigra rescues motor activity and neuronal cell death in PD-induced mouse models.
• DX2 inhibits TRAF2 dependent and 67 LR mediated neuronal death in ALS disease.
• Overexpression of DX2 improved locomotive activity and survival and reduced neuronal cell death in the ALS mouse model.
• Adeno-associated virus has emerged as the preferred vector for targeting gene expression to the retina. Subretinally or by intra vitreally injected AAV can efficiently transduce retinal pigment epithelium and inner layer of retina. AAV is also appreciated as a suitable gene delivery approach because of its relative safety due to a lack of pathogenicity (20). Utilization of survival gene DX2 can be a potential option for treatment of retinal degenerative diseases.
• AIMP2-DX2 is an alternative, antagonistic splicing variant of AIMP2 (aminoacyl tRNA synthase complex-interacting multifunctional protein 2), which is a multifactorial apoptotic gene.
• AIMP2-DX2 is known to suppress cell apoptosis by hindering the functions of AIMP2.
• AIMP2-DX2 acting as a competitive inhibitor of AIMP2, suppresses TNF-alpha mediated apoptosis through inhibition of ubiquitination/degradation of TRAF2.
• AIMP2-DX2 can treat neuronal diseases (US2019/0298858 Al).
• nucleic acid molecule comprising an exon 2-deleted AIMP2 variant (AIMP2-DX2 or DX2).
• the nucleic acid molecule is a viral vector or a nonviral vector, e.g., a recombinant vector.
• a recombinant vector comprising an exon 2-deleted AIMP2 variant (AIMP2-DX2 or DX2) gene.
• the retinal degenerative disease is retinitis pigmentosa, Leber’s congenital amaurosis, Cone -rod dystrophy, glaucoma, or diabetic retinopathy. In some embodiments, the retinal degenerative disease precedes or is accompanied by Parkinson’s disease, Alzheimers’s disease, or amyotrophic lateral sclerosis. In some embodiments, the retinal degenerative disease is not age-related macular disease. In some embodiments, the methods of treating retinal degenerative diseases does not include treating an age-related macular disease. In some embodiments, the AMD is wet AMD. In some embodiments, the AMD is dry AMD.
• AIMP2-DX2 exon 2-deleted AIMP2 variant
• the recombinant vectors as disclosed herein can further comprise an miR-142 target sequence.
• the vector can further comprise a promoter operably linked to the AIMP2-DX2.
• the promoter is a Retrovirus (LTR) promoter, cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, MT promoter, EF-1 alpha promoter, UB6 promoter, chicken beta-actin promoter, CAG promoter, RPE65 promoter, Synapsin promoter, MeCP2 promoter, CaMKII promoter, Hb9 promoter, or opsin promoter.
• the recombinant vectors can comprise exon 2-deleted AIMP2 variant (AIMP2-DX2) gene and an miR-142 target sequence.
• the miR-142 target sequence can be 3’ to the AIMP2-DX2 gene.
• the vectors described herein can express AIMP2-DX2 in neuronal cells but not in hematopoietic cells, such as leukocytes and lymphoid cells.
• the AIMP2-DX2 polypeptide (SEQ ID NO:2) is a splice variant of AIMP2 (e.g., aa sequence of SEQ ID NO: 12; e,g., nt sequence of SEQ ID NOG), in which the second exon (SEQ ID NO: 10; nt sequence of SEQ ID NO:4) of AIMP2 is omitted.
• the AIMP2- DX2 gene has a nucleotide sequence set forth in SEQ ID NO: 1
• the AIMP2-DX2 polypeptide has an amino acid sequence set forth in SEQ ID NO:2.
• FIGS. 6A-6C show a comparison of AIMP2 (SEQ ID NO:2) and variants, SEQ ID NOS: 13-19, as well as a consensus or core sequence of AIMP2 or AIMP2-DX2 (SEQ ID NO:20).
• the AIMP2-DX2 gene can comprise a nucleotide sequence encoding an amino acid sequence that is at least 90% identical, at least 93% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical to SEQ ID NO:2, 13, 14, 15, 16, 17, 18, 19, or 20, or any ranges of % identity therein.
• the AIMP2-DX2 gene can comprise a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:2, 13, 14, 15, 16, 17, 18, 19, or 20.
• the AIMP2-DX2 gene can comprise a nucleotide sequence at least 90% identical, at least 93% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical to a nucleotide sequence of SEQ ID NO: 1, or any ranges of % identity therein.
• the AIMP2-DX2 gene can comprise a nucleotide sequence of SEQ ID NO: 1.
• the AIMP2-DX2 gene does not have an exon comprising a nucleotide sequence encoding an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOTO or 11.
• the AIMP2-DX2 gene does not have an exon comprising a nucleotide sequence encoding an amino acid sequence of SEQ ID NOTO or 11.
• the AIMP2-DX2 gene does not have an exon comprising a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOT.
• the nucleic acid molecule comprising AIMP2-DX2 can optionally comprise an miR-142 target sequence.
• the miR-142 target sequence (miR-142T) can comprise a nucleotide sequence comprising AC ACTA.
• the miR-142 target sequence can comprise a nucleotide sequence comprising AC ACTA and 1-17 additional contiguous nucleotides of SEQ ID NO:5.
• the miR-142 target sequence can comprise a nucleotide sequence comprising ACACTA and a sum of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 additional nucleotides that are contiguous 5’ or 3’ of ACACTA as shown in SEQ ID NO:5.
• the miR-142 target sequence can comprise a nucleotide sequence at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, at least 90% identical, at least 93% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to a nucleotide sequence of SEQ ID NO: 5 (TCCATAAAGTAGGAAACACTACA; miR-142-3pT).
• the miR-142 target sequence can comprise a nucleotide sequence of SEQ ID NO:5.
• the miR-142 target sequence can comprise a nucleotide sequence comprising ACTTTA.
• the miR-142 target sequence can comprise a nucleotide sequence comprising ACTTTA and 1-15 additional contiguous nucleotides of SEQ ID NOT.
• the miR-142 target sequence can comprise a nucleotide sequence comprising ACTTTA and a sum of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additional nucleotides that are contiguous 5’ or 3’ of ACTTTA as shown in SEQ ID NOT.
• the miR-142 target sequence can comprise a nucleotide sequence at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, at least 90% identical, at least 93% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to a nucleotide sequence of SEQ ID NO:7 (AGTAGTGCTTTCTACTTTATG; miR-142-5pT).
• the miR-142 target sequence can comprise a nucleotide sequence of SEQ ID NO:7.
• An example miR142-3pT mutant sequence is: ccgctgcagtgtgacagtgccagccaatgtgcagaggtggatgaggtcttgtgaaaacctggctcctttaacacggccctcaagct ccttaagtgaccagaagcttgctagctccataaagtaggaCCACTGCAatcactccataaagtaggaCCACTGCAagatatct ccataaagtaggaCCACTGCAatcactccataaagtaggaCCACTGCAatcactccataaagtaggaCCACTGCAaaagcttgtagggatccgcc (SEQ ID NO:25).
• a mutant sequence refers to one or more regions, e.g., four regions of core sequences of miR142 3pT that are substituted as follows: (5’- AACACTAC-3’ 5’-CCACTGCA-3’).
• Inhibition of DX2 expression in vector transfected HEK293 cells was observed with the miR142- 3p xl repeat (100 pmol) miR142-3p target sequence and as the number of core binding sequence in miR142-3p target seq are increased, miR142-3p inhibition on DX2 expression was also increased.
• the Tseq x3 core sequence containing vector showed significant inhibition, whereas no inhibition was observed for the mutated 3x sequence.
• a microRNA is a non-coding RNA molecule that functions to control gene expression.
• MiRNAs function via base-pairing with complementary sequences within mRNA molecules, i.e., a miRNA target sequences.
• miRNAs can bind to target messenger RNA (mRNA) transcripts of protein-coding genes and negatively control their translation or cause mRNA degradation.
• mRNA target messenger RNA
• miRbase databases are publicly available. Many miRNAs are expressed in a tissue-specific manner and have an important roles in maintaining tissue-specific functions and differentiation.
• MiRNA acts at the post-transcription stage of the gene and, in the case of mammals, and it is known that approximately 60% of the gene expression is controlled by miRNA.
• miRNA plays an important role in a diverse range of processes within a living body and has been disclosed to have correlation with cancer, cardiac disorders and nerve related disorders.
• miR- 142-3p and miR-142-5p exist in miR-142 and any of the target sequences thereof can be used.
• miR-142 or “miRNA-142” refers to, e.g., miR-142-3p and/or miR-142-5p, and can bind to the miR-142 target sequence, e.g., miR-142-3pT or miR-142-5pT.
• the miR-142 target sequence can be 5’ or 3’ to the AIMP2-DX2 gene.
• miR-142-3p can exist in the area at which translocation of its gene occurs in aggressive B cell leukemia and is known to express in hemopoietic tissues (bone marrow, spleen and thymus, etc.).
• miR-142-3p is known to be involved in the differentiation of hemopoietic system with confirmation of expression in the liver of fetal mouse (hemopoietic tissue of mouse).
• the miR-142-3p and/or miR-142-5p target sequence is repeated at least 2-10 times, at least 2-8 times, at least 2-6 times, at least 4 times, or any range or number of times thereof.
• the miR-142-3p e.g., having a nucleotide sequence of SEQ ID NO:23
• a corresponding target sequence e.g., a miR-142-3p target sequence (miR-142-3pT) having a nucleotide sequence of SEQ ID NO:5 but not limited thereto.
• the miR-142-5p e.g., having a nucleotide sequence of SEQ ID NO:24 can have a corresponding target sequence, e.g., a miR-142- 5p target sequence (miR-142-5pT) having a nucleotide sequence of SEQ ID NO:7 but not limited thereto.
• an miR-142-3p can have a nucleotide sequence of SEQ ID NO:23 and an miR-142-5p can have a nucleotide sequence of SEQ ID NO: 24.
• recombinant vectors that can control the side effect of over-expression of the AIMP2-DX2 variant by inserting an miR-142-3p target sequence and/or miR-142-5p target sequence (miR-142-3pT and/or miR-142-5pT, respectively) into a terminal end of AIMP2-DX2 and controlling suppression of AIMP2-DX2 expression in CD45-derived cells, in particular, the lymphatic system and leukocytes.
• the expression of AIMP2-DX2 variant can be restricted to only in the injected neuronal cells and tissues but not in non-neuronal hematopoietic cells, the major population in the injected tissue areas.
• MiR142-3p is expressed only in hematopoietic cells.
• Disclosed herein are recombinant vectors containing a target sequence for miR-142-3p and/or miR-142-5p.
• Disclosed herein are recombinant vectors comprising an exon 2-deleted AIMP2 variant (AIMP2-DX2) gene and miR-142-3p and/or miR-142-5p target sequences as disclosed herein.
• the term “recombinant vector” refers to a vector that can encode a target protein or RNA in appropriate host cells, or a gene construct that contains essential operably linked control factor(s) to enable the inserted gene to be expressed appropriately.
• the term “operably linked” refers to functional linkage between the nucleic acid expression control sequence and nucleic acid sequence that codes the targeted protein and RNA to execute general functions. For example, it can affect the expression of nucleic acid sequence that codes promoter and protein or RNA that has been linked for operability of the nucleic acid sequence. Operable linkage with recombinant vector can be manufactured by using gene recombinant technology, which is known well in the corresponding technology area, and uses generally known enzymes in the corresponding technology area for the area-specific DNA cutting and linkage.
• the recombinant vectors can further comprise a promoter operably linked to a AIMP2- DX2 as disclosed herein.
• the promoter is a Retrovirus (LTR) promoter, cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, MT promoter, EF-1 alpha promoter, UB6 promoter, chicken beta-actin promoter, CAG promoter, RPE65 promoter, Synapsin promoter, MeCP2 promoter, CaMKII promoter, Hb9 promoter, or opsin promoter.
• the recombinant vector can additionally contain heterogeneous promoter and operably linked heterogeneous gene in the promoter.
• Heterogeneous gene as used herein can include protein or polypeptide with biologically appropriate activation, and encrypted sequence of the targeted product such as immunogen or antigenic protein or polypeptide, or treatment activation protein or polypeptide.
• Polypeptides can supplement deficiency or absent expression of endogenous protein in host cells.
• the gene sequence can be induced from a diverse range of suppliers including DNA, cDNA, synthesized DNA, RNA or its combinations.
• the gene sequence can include genome DNA that contains or does not contain a natural intron.
• the genome DNA can be acquired along with promoter sequence or polyadenylated sequence.
• Genome DNA or cDNA can be acquired in various methods. Genome DNA can be extracted and purified from appropriate cells through methods publicly notified in the corresponding area. Alternatively, mRNA can be used to produce cDNA by reverse transcription or other methods by being separated from the cells.
• polynucleotide sequence can contain sequence that is complementary to RNA sequence, e.g., antisense RNA sequence, and the antisense RNA can be administered to individual to suppress expression of complementary polynucleotide in the cells of individuals.
• the heterogeneous gene is an AIMP-2 splicing variant with the loss of exon 2 and miR-142-3p target sequence can be linked to 3’ UTR of the heterogeneous gene.
• the sequence of the AIMP2 protein (312aa version: AAC50391.1 or GI: 1215669; 320aa version: AAH13630.1, GI: 15489023, BCO 13630.1) are described in the literature (312aa version: Nicolaides, N.C., Kinzler, K.W. and Vogelstein, B.
• AIMP2 splicing variant refers to the variant generated due to partial or total loss of exon 2 among exons 1 to 4. As such, the variant signifies interference of the normal function of AIMP2 by forming AIMP2 protein and heterodimer.
• the injected AIMP2-DX2 gene is rarely expressed in tissues other than the injected tissue.
• an miR142 target sequence can be inserted to completely block the possibility of AIMP2-DX2 being expressed in hematopoietic cells, the major population of non-neuronal cells in the injected tissue area.
• the recombinant vector can include SEQ ID NOS:! and 5.
• % of sequence homology can be, e.g., confirmed by comparing the 2 optimally arranged sequence with the comparison domain and some of the nucleotide sequences in the comparison domain can include addition or deletion (that is, gap) in comparison to the reference sequence on the optimal arrange of the 2 sequences (does not include addition or deletion).
• Proteins as disclosed herein not only include those with its natural type amino acid sequence but also those with variant amino acid sequences.
• Variants of the protein signifies proteins with difference sequences due to the deletion, insertion, non-conservative or conservative substitution or their combinations of the natural amino acid sequence and more than 1 amino acid residue. Amino acid exchange in protein and peptide that does not modify the activation of the molecule in overall is notified in the corresponding area (H.Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979).
• the protein or its variant can be manufactured through natural extraction, synthesis (Merrifield, J. Amer. Chem. Soc. 85: 2149-2156, 1963), or genetic recombination on the basis of the DNA sequence (Sambrook et al, Molecular Cloning, Cold Spring Harbour Laboratory Press, New York, USA, 2 nd Ed., 1989).
• Amino acid mutations can occur on the basis of the relative similarity of the amino acid side chain substituent such as hydrophilicity, hydrophobicity, electric charge and size, etc.
• amino acid side chain substituent such as hydrophilicity, hydrophobicity, electric charge and size, etc.
• arginine, lysine and histidine are residues with positive charge
• alanine, glycine and serine have similar sizes
• phenylalanine, tryptophan and tyrosine have similar shapes.
• arginine, lysine and histidine alanine, glycine and serine
• phenylalanine, tryptophan and tyrosine can be deemed functional equivalents biologically.
• hydrophobic index of amino acid can be considered. Hydrophobic index is assigned to each amino acid according to hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (- 1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5)
• hydrophobic amino acid index is very important. It is possible to have similar biological activation only if a substitution is made with an amino acid with a similar hydrophobic index. In the event of introducing a mutation by making reference to the hydrophobic index, substitution between amino acids with hydrophobic index differences within ⁇ 2, within ⁇ 1, or within ⁇ 0.5.
• substitutions can be made between amino acids with hydrophilic value differences within ⁇ 2, within ⁇ 1, or within ⁇ 0.5. but not limited thereto.
• Vectors disclosed herein can be constructed as a typical vector for cloning or for expression.
• the vectors can be constructed with prokaryotic or eukaryotic cells as the host. If the vector is an expression vector and prokaryotic cell is used as the host, it is general to include powerful promoter for execution of transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, Ipp promoter, pL X promoter, pRX promoter, rac5 promoter, amp promoter, rec A promoter, SP6 promoter, trp promoter and T7 promoter, etc.), ribosome binding site for commencement of decoding and transcription/decoding termination sequence.
• powerful promoter for execution of transcription e.g., tac promoter, lac promoter, lacUV5 promoter, Ipp promoter, pL X promoter, pRX promoter, rac5 promoter, amp promoter, rec A promoter, SP6 promoter, trp promoter and T7 promoter, etc
• coli e.g., HB101, BL21, DH5a, etc.
• promoter and operator site of the tryptophan biosynthesis route of E. coli (Yanofsky, C. (1984), J. Bacterio!., 158: 1018-1024) and left directional promoter of phage X (pLX promoter, Herskowitz, I. and Hagen, D. (1980), Ann. Rev. Genet., 14: 399-445) can be used as the control site.
• vectors that can be used can be more than 1 type, such as a virus vector, linear DNA, or plasmid DNA.
• Virus vector refers to a virus vector capable of delivering gene or genetic substance to the desired cells, tissue and/or organ.
• the virus vectors can include more than 1 species from the group composed of Adenovirus, Adeno-associated virus, Lentivirus, Retrovirus, HIV (Human immunodeficiency virus), MLV (Murine leukemia virus), ASLV (Avian sarcoma/leukosis), SNV (Spleen necrosis virus), RSV (Rous sarcoma virus), MMTV (Mouse mammary tumor virus) and Herpes simplex virus, it is not limited thereto.
• the viral vector can be an adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, vaccinia virus, or herpes simplex virus vector.
• Retrovirus Although Retrovirus has an integration function for the genome of host cells and is harmless to the human body, it can have characteristic including suppressing functions of normal cells at the time of integration, ability to infect a diverse range of cells, ease of proliferation, accommodate approximately 1-7 kb of external gene, and generate duplication deficient virus.
• Retroviruses can also have disadvantages including difficulties in infecting cells after mitotic division, gene delivery under an in vivo condition, and need to proliferate somatic cells under in vitro condition.
• Retroviruses have the risk of spontaneous mutations as it can be integrated into proto-oncogene, thereby presenting the possibility of cell necrosis.
• Adenoviruses have various advantages as a cloning vector including duplication even in nucleus of cells in medium level size, clinically nontoxic, stable even if external gene is inserted, no rearrangement or loss of genes, transformation of eukaryotic organism and stably undergoes expression at high level even when integrated into host cell chromosome.
• Good host cells of Adenoviruses are the cells that are the causes of hemopoietic, lymphatic and myeloma in humans.
• proliferation is difficult since it is a linear DNA and it is not easy to recover the infected virus along with low infection rate of virus.
• expression of the delivered gene is most extensive during 1-2 weeks with expression sustained over the 3-4 weeks only in some of the cells. Another issue is that it has high immuno-antigenicity.
• Adeno-associated virus has been preferred in recent years since it can supplement the aforementioned problems and has a lot of advantages as gene therapy agent. It is also referred to as adeno-satellite virus. The diameter of adeno-associated virus particle is 20nm and is known to have almost no harm to the human body. As such, its sales as gene therapy agent in Europe were approved.
• AAV is a provirus with single strand that needs auxiliary virus for duplication and AAV genome has 4,680 bp that can be inserted into specific area of the chromosome 19 of the infected cells.
• Trans-gene is inserted into the plasma DNA connected by the 2 inverted terminal repeat (ITR) sequence section with 145bp each and signal sequence section.
• ITR 2 inverted terminal repeat
• Transfection is executed along with other plasmid DNA that expresses the AAV rep and cap sections, and Adenovirus is added as an auxiliary virus.
• AAV has the advantages of a wide range of host cells that deliver genes, little immunological side effects at the time of repetitive administration and long gene expression period. Moreover, it is safe even if the AAV genome is integrated with the chromosome of host cells and does not modify or rearrange the gene expression of the host.
• the Adeno-associated virus is known to have a total of 4 serotypes.
• the most widely researched vector is the Adeno-associated virus serotype 2 and is currently used in the delivery of clinical genes of cystic fibrosis, hemophilia and Canavan’s disease.
• the potential of recombinant adeno-associated virus (rAAV) is increasing in the area of cancer gene therapy (Du 2013).
• the Adeno-associated virus serotype 2 can be used. Although it is possible to select and apply appropriate viral vector, it is not limited to this.
• vectors are expression vectors and use eukaryotic cells as the host
• promoter derived from the genome of mammalian cells e.g., metallothionein promoter
• promoter derived from mammalian virus e.g., post-adenovirus promoter, vaccine virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and HSV TK promoter
• telomere a virus that promotes the transcription termination sequence.
• CMV cytomegalovirus
• RSV Rous sarcoma virus
• MT MT promoter
• EF-1 alpha promoter a promoter that promotes the transcription termination sequence.
• UB6 Rous sarcoma virus
• UB6 EF-1 alpha promoter
• UB6 EF-1 alpha promoter
• UB6 EF-1 alpha promoter
• UB6 promoter EF-1 alpha promoter
• UB6 promoter EF-1 alpha promoter
• UB6 promoter EF-1 alpha promoter
• UB6 promoter EF-1 alpha promoter
• UB6 promoter EF-1 alpha promoter
• UB6 promoter EF-1 alpha promoter
• UB6 promoter EF-1 alpha promoter
• UB6 promoter EF-1 alpha promoter
• UB6 promoter EF-1 alpha promoter
• UB6 promoter EF-1 alpha promote
• Vectors disclosed herein can be fused with other sequences as needed to make the purification of the protein easier.
• a fused sequence such as glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6xHis (hexahistidine; Qiagen, USA), etc.
• expression vectors can include tolerance gene against antibiotics generally used in the corresponding industry as the selective marker including Ampicillin, Gentamycin, Carbenicillin, Chloramphenicol, Streptomycin, Kanamycin, Geneticin, Neomycin and Tetracycline, as examples.
• gene carriers including the recombinant vector containing a target sequence (miR-142-3pT and/or miR-142-5pT) for miR-142, such as miR-142-3p and/or miR-142-5p, respectively.
• the term “gene transfer” includes delivery of genetic substances to cells for transcription and expression in general. Its method is ideal for protein expression and treatment purposes. A diverse range of delivery methods such as DNA transfection and virus transduction are announced. It signifies virus-mediated gene transfer due to the possibility of targeting specific receptor and/or cell types through high delivery efficiency and high level of expression of delivered genes, and, if necessary, nature-friendliness or pseudo-typing.
• the gene carriers can be transformed entity that has been transformed into the recombinant vector, and transformation includes all methods of introducing nucleic acid to organic entity, cells, tissues or organs and as announced in the corresponding area, it is possible to select and execute appropriate standard technology in accordance with the host cells. Although such methods include electroporation, fusion of protoplasm, calcium phosphate (CaPfh) sedimentation, calcium chloride (CaCh) sedimentation, mixing with the use of silicone carbide fiber, agribacteria-mediated transformation, PEG, dextran sulphate and lipofectamin, etc., it is not limited to these.
• the gene carriers are for the purpose of expression of heterogeneous genes in neuron. As such it suppresses the expression of the heterogeneous gene in CD45-derived cells and can increase the expression of heterogeneous gene in brain tissue.
• the majority of the CD45 are transmembrane protein tyrosine phosphatase situated at the hematopoietic cell. Cells can be defined in accordance with the molecules situated on the cell surface and CD45 is the cell marker for all leukocyte groups and B lymphocytes.
• the gene carrier is not expressed in the CD45-derived cells, in particular, in lymphoid and leukocyte range of cells.
• the gene carriers can additionally include carrier, excipient or diluent allowed to be used pharmacologically.
• the methods of delivery disclosed herein can increase the expression of heterogeneous gene in cerebral tissues and control heterogeneous gene expression in other tissues.
• vectors comprising 1) a promoter; 2) a nucleotide sequence that encodes a target protein linked with the promoter to enable operation; and 3) an expression cassette that includes the nucleotide sequence targeting miR-142-3p inserted into 3’UTR of the nucleotide sequence.
• the vectors can comprise 1) a promoter; 2) a nucleotide sequence that encodes a target protein linked with the promoter to enable operation; and 3) an expression cassette that includes the nucleotide sequence targeting miR-142-5p inserted into 3’UTR of the nucleotide sequence.
• expression cassette refers to the unit cassette that can execute expression for the production and secretion of the target protein operably linked with the downstream of signal peptide as it includes gene that encodes the target protein and nucleotide sequence that encodes the promoter and signal peptide.
• Secretion expression cassette can be used mixed with the secretion system. A diverse range of factors that can assist the efficient production of the target protein can be included in and out of such expression cassette.
• preventive or therapeutic preparations for retinal degenerative disease that includes a nucleotide sequence that encodes AIMP-2 splicing variant with loss of exon 2 and nucleotide sequence that targets miR-142-3p linked to 3’UTR of the nucleotide sequence.
• the retinal degenerative disease does not include AMD.
• AMD can be is wet AMD.
• the AMD is dry AMD.
• the vectors disclosed herein can affect, but not limited to, apoptosis inhibition, dyskinesia amelioration, and/or oxidative stress inhibition, and thus prevent or treat retinal degenerative disease.
• treatment includes not only complete treatment of retinal degenerative disease but also partial treatment, improvement and/or reduction in the overall symptoms of AMD as results of application of the pharmacological agent disclosed herein.
• prevention signifies prevention of the occurrence of overall symptoms of retinal degenerative disease in advance by suppressing or blocking the symptoms or phenomenon such as cognition disorder, behavior disorder and destruction of brain nerves by applying pharmacological agents disclosed herein to the entity with degenerative cerebral disorders.
• Adjuvants other than the active ingredients can be included additionally to the pharmacological agents disclosed herein. Although any adjuvant can be used without restrictions as long as it is known in the corresponding technical area, it is possible to increase immunity by further including complete and incomplete adjuvant of Freund, for example.
• Pharmacological agents disclosed herein can be manufactured in the format of having mixed the active ingredients with the pharmacologically allowed carrier.
• pharmacologically allowed carrier includes carrier, excipient and diluent generally used in the area of pharmacology.
• Pharmacologically allowed carrier that can be used for the pharmacological agents disclosed herein include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil, but not limited to these.
• Pharmacological agents disclosed herein can be used by being manufactured in various formats including oral administration types such as powder, granule, pill, capsule, suspended solution, emulsion, syrup and aerosol, etc., and external application, suppository drug or disinfection injection solution, etc. in accordance with their respective general manufacturing methods.
• Solid preparations for oral administration include pill, tablet, powder, granule and capsule preparations, and such solid preparations can be manufactured by mixing more than 1 excipient such as starch, calcium carbonate, sucrose, lactose and gelatin with the active ingredients.
• lubricants such as magnesium stearate and talc can also be used in addition to simple excipients.
• Liquid preparations for oral administration include suspended solution, solution for internal use, oil and syrup, etc.
• Preparations for non-oral administration include sterilized aqueous solution, non-aqueous solvent, suspension agent, oil, freeze dried agent and suppository. Vegetable oil such as propylene glycol, polyethylene glycol and olive oil, and injectable esters such as ethylate can be used as non-aqueous solvent and suspension solution.
• Agents for suppository can include witepsol, tween 61, cacao oil, laurine oil and glycerogelatin, etc.
• Pharmacological agents disclosed herein can be administered into entity through diversified channels. All formats of administration such as oral administration, and intravenous, muscle, subcutaneous and intraperitoneal injection can be used.
• the recombinant vector is administered topically to, by intravitreal injection to, by subconjunctival injection to, or into a subretinal space of the subject.
• the methods disclosed herein can further comprise administering to the subject an additional therapeutic agent(s).
• the additional therapeutic agent is ranibizumab, aflibercept, and/or bevacizumab.
• Desirable doses of administration of therapeutic agents disclosed herein differs depending on various factors including preparation production method, administration format, age, weight and gender of the patient, extent of the symptoms of the disease, food, administration period, administration route, discharge speed and reaction sensitivity, etc. Nonetheless, it can be selected appropriately by the corresponding manufacturer.
• the skilled medical doctor can determine and prescribe effective dose for the targeted treatment.
• the treatment routes can include but are not limited to subretinal, intravitreal, intravenous, subcutaneous and muscle injection, and direction injection into the eye, cerebral ventricle or spinal cord by using micro-needle.
• the dose per eye can be IxlO 8 vg (viral genome) to IxlO 11 vg, IxlO 8 vg to IxlO 10 vg, IxlO 8 vg to IxlO 9 vg, and any specific doses or ranges of doses therein, e.g., 2xl0 8 vg to 5xl0 8 vg, 2xl0 9 vg to 4xlO 10 vg, 4xl0 8 vg to 6xlO 10 vg, 5xl0 8 vg to 4xlO 10 vg, 5xl0 8 vg to IxlO 11 vg, IxlO 9 vg to 5xlO 10 vg, 2xl0 9 vg to IxlO 10 vg, 5xl0 9 vg to IxlO 10 vg, or 5xl0 9 vg to 8xlO 10 vg.
• the effective dose can be, e.g., per eye, 0.05 to 15 mg/kg in the case of vector, 5xl0 n to 3.3xl0 14 viral particle (2.5xl0 12 to 1.5xl0 16 IU)/kg in the case of recombinant virus and 5xl0 2 to 5xl0 7 cells/kg in the cells.
• the doses are 0.1 to 10 mg/kg in the case of vector, 5xl0 12 to 3.3xl0 13 particles (2.5xl0 13 to 1.5xl0 15 IU)/kg in the case of recombinant virus and 5xl0 3 to 5xl0 6 cells/kg in the case of cells at the rate of 2 to 3 administrations per week.
• the dose is not strictly restricted. Rather, it can be modified in accordance with the condition of the patient and the extent of manifestation of the neural disorders.
• Effective dose for other subcutaneous fat and muscle injection, and direct administration into the affected area is 9xlO 10 to 3.3xl0 14 recombinant viral particles with the interval of 10cm and at the rate of 2-3 times per week. The dose is not strictly restricted.
• pharmacological agents disclosed herein can include IxlO 10 to IxlO 12 vg (virus genome )/mL of recombinant adeno-associated virus and, generally, it is advisable to inject IxlO 12 vg once every 2 days over 2 weeks. It can be administered once a day or by dividing the dose for several administrations throughout the day.
• the vectors can be administered in a dose of O.lxlO 8 vg to 500xl0 8 vg, IxlO 8 vg to 100xl0 8 vg, IxlO 8 vg to 10xl0 8 vg, e.g., 5xl0 8 vg, or any specific amounts or ranges derived therefrom.
• IV injections e.g., vg can be translated to doses for humans based on body weight for IV injection.
• vg can also be translated to doses for humans based on the target cell number and effective MOI (multiplicity of infection).
• the vectors disclosed herein can be injected to a subject by, e.g., subretinal injection, intravitreal injection, or subchoroidal injection.
• the injection can be in the form of a liquid.
• the vectors disclosed herein can be administered to a subject in the form of eye drops or ointment.
• nucleic acid molecules comprising AIMP2-DX2 can be delivered by methods well known in the art.
• the nucleic acid molecules comprising AIMP2-DX2 can also be optionally combined with other gene or nucleic acids delivery tools or materials.
• Delivery methods can include but are not limited to: viral vectors and non- viral vectors; chemical conjugation with Galnac, cell-penetrating peptides, nucleic acids or glycopeptide mediated tools, or synthetic compound vectors; lipid-mediated delivery such as lipid nanoparticle encapsulation; inorganic vector mediated delivery such as inorganic vectors; biological delivery such as exosome mediated delivery; physical delivery such as by micro/nano needles, pressure -perfusion, microprojectiles, electrical energy/electroporation/iontophoresis, sonoporation, magnetoporation, or optoporation/photodynamic energy.
• the pharmacological preparations can be produced in a diverse range of orally and non- orally administrable formats.
• the vector disclosed herein can be administered to the brain or spinal cord.
• the vectors disclosed herein can be administered to the brain by stereotaxic injection.
• Orally administrative agents include pills, tablets, hard and soft capsules, liquid, suspended solution, oils, syrup and granules, etc. These agents can include diluent (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine) and glidents (e.g., silica, talc, and stearic acid and its magnesium or calcium salts, and/ or polyethylene glycol) in addition to the active ingredients.
• diluent e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine
• glidents e.g., silica, talc, and stearic acid and its magnesium or calcium salts, and/ or polyethylene glycol
• the pills can contain binding agents such as magnesium aluminum silicate, starch paste, gelatin, tragacanthin, methyl cellulose, sodium carboxymethyl cellulose and/or polyvinyl pyrrolidine, and, depending on the situation, can contain disintegration agent such as starch, agar, alginic acid or its sodium salt or similar mixture and/or absorbent, coloring, flavor and sweetener.
• the agents can be manufactured by general mixing, granulation or coating methods.
• injection agents are the representative form of non-orally administered preparations. Solvents for such injection agents include water, Ringer’s solution, isotonic physiological saline and suspension.
• Sterilized fixation oil of the injection agent can be used as solvent or suspension medium, and any non-irritating fixation oil including mono- and di-glyceride can be used for such purpose.
• the injection agent can use fatty acids such as oleic acid.
• CD45 transmembrane protein tyrosine phosphatase of the hematopoietic cell, which can be used to define the cells in accordance with the molecule on the cell surface.
• CD45 is a marker for all leukocyte groups and B lymphocytes.
• a recombinant vector has been produced that is expressed specifically and only in neurons without being expressed in CD45-derived cells, in particular, lymphoid and leukocyte cells.
• the recombinant vector contains a splicing variant in which exon 2 of the Aminoacyl tRNA Synthetase Complex Interacting Multifunctional Protein 2 (AIMP2) has been deleted and an miRNA capable of controlling the expression of the AIMP2 splicing variant.
• AIMP2 Aminoacyl tRNA Synthetase Complex Interacting Multifunctional Protein 2
• the recombinant vector was produced as a distribution safety measure in order to induce specific expression of the AIMP2 splicing variant in injected neuronal tissues. Also this was done to completely block any possibility of AIMP2-DX2 being expressed in hematopoietic cells, which is the major population of non-neuronal cells in the injected tissue area.
• AIMP2 is one of the proteins involved in the formation of aminoacyl-tRNA synthetase (ARSs) and acts as a multifactorial apoptotic protein.
• ARSs aminoacyl-tRNA synthetase
• cDNA of AIMP2 splicing variant was cloned into pcDNA3.1-myc.
• the sub-cloning in pcDNA3.1-myc was executed by using EcoRl and Xho 1 after having amplified the AIMP2 splicing variant by using a primer having EcoR 1 and Xho 1 linker attached to the H322 cDNA.
• AIMP2 variant having a nucleotide sequence of SEQ ID NO: 1 and an amino acid sequence of SEQ ID NO:2 was used.
• the recombinant vector was produced as above in order to confine the expression of the AIMP2 variant in injected neuronal cells and to completely block the possibility of AIMP2-DX2 being expressed in hematopoietic cells, the major population of non-neuronal cells in the injected tissue area.
• miR-142-3p that is specifically expressed only in hematopoietic cells that generate leukocyte and lymphoid related cells was selected as the target.
• miR-142-3p In order to produce the sequence that targets only the miR-142-3p, microarray data of mouse B cells and computer programming of genes targeted by miRT42-3p (mirSVR score) were used.
• the miR-142-3p is a nucleotide sequence indicated SEQ ID NO.
• the miR-142-3p target sequence of SEQ ID NO:5 binds to miR-142-3p.
• the miR-142-3p target sequence includes Nhe 1 and Hind III, Bmt 1 site sequence (ccagaagcttgctagc; SEQ ID NO:21) and Hind H site sequence (aagcttgtag; SEQ ID NO:22).
• the miR-142-3pT can comprise the nucleotide sequence of SEQ ID NO:5 that has been repeated 4 times with the linkers (tcac and gatatc) that connects them (FIG. 4; SEQ ID NO:6).
• Example 1-3 Production of the recombinant vector
• the miR-142-3p target sequence (SEQ ID NO:5) was inserted into 3’UTR of the AIMP2 variant (sequence number of 1). Connecting of the AIMP-2 variant and miR-142-3p target sequence is indicated with base sequence number of 6, and, specifically, was cut and inserted by using Nhe I and Hind III sites.
• the recombinant vector is shown in FIG. 1.
• Example 1 -4 Confirmation of the nerve cells specific expression of recombinant vector in vitro [0169] Since miR142-3p is specifically expressed only in hemopoietic cells, the extent of the expression of AIMP2 variant was confirmed in specific cells in accordance with the knockdown of AIMP2 variant according to the expression of miR142-3p target sequence of the recombinant vector.
• AIMP2 variant is not expressed in the SHAM and NC vector groups.
• the AIMP2 variant is specifically expressed only in the SH-SY5Y cell strain in the group treated with the recombinant vector (FIG. 2).
• Example 1-5-2. miR142-3p inhibition experiment [0173] miR-142-3p inhibition on DX2 expression could be observed from xl miR-142-3p target sequence.
• the HEK293 cells were transiently transfected with the xl, x2, and x3 repeat miR-142- 3p target sequence vectors, and also with 100 pmol miR-142-3p using lipofectamine 2000 (Invitrogen, US), and then incubated for 48 hrs. The amount of DX2 mRNA was analyzed by PCR. miR142-3p inhibition on DX2 expression was observed from Tseq xl repeat miR142-3p target seq (FIG. 4B).
• Tseq xl contains 1 core binding sequence
• Tseq x2 contains 2 core binding sequences
• Tseq x3 contains 3 core binding sequences (FIG. 4A).
• miR142-3p (100 pmol) inhibition on DX2 expression was started to be observed from xl repeat miR142-3p target sequence.
• the HEK293 cells were transiently transfected with the xl, x2, and x3 repeat miR-142-3p T seq vectors, and also with 100 pmol miR-142-3p using lipofectamin 2000 (invitrogen, US), then incubated for 48 h. Amount of DX2 mRNA was analyzed by PCR. When the number of core binding sequence in miR142-3p target seq are increased, miR142-3p inhibition on DX2 expression was also increased. Tseq x3 core sequence containing vector showed significant inhibition (FIG. 4B).
• Example 1-7 Core sequence mutation.
• Example 1-8 Core binding sequence is important DX2 inhibition
• FIG. 4A Four core sequences were substituted (FIG. 4A).
• the HEK293 cells were transiently transfected with the DX2- miR-142-3p T seq x3 repeated vector (Tseq3x) or with core sequence mutated vector (mut), and with 100 pmol miR-142-3p by using lipofectamin 2000 (Invitrogen, US), and then incubated for 48 hrs.
• Expression of DX2 mRNA was analyzed by PCR.
• Tseq x3 repeated vector which showed significant inhibition of DX2 (FIG. 4B) and DX2 construct were used as control.
• 100 pmol of miR142-3p treatment inhibited Tseq x3 vector significantly but DX2 and mut sequence were not inhibited (FIG. 5).
• AIMP2 was considered as a target for inhibiting cell death.
• AIMP2 accumulation leads to poly (ADP-ribose) polymerase- 1 (PARP-1) activation and subsequent degeneration of neurons: DX2 compromises the pro-apoptotic activity of AIMP2 via the competitive inhibition of AIMP2 binding to PARP-1.
• PARP-1 poly (ADP-ribose) polymerase- 1
• PARP-1 The enzyme poly (ADP-ribose) polymerase- 1 (PARP-1) is the most studied member of the PARP family (also called ADPRT, PARS, ART), which comprises 17 members responsible for poly (ADP-ribose) (PAR) synthesis.
• ADPRT ADPRT
• PARS ART
• PARP enzymes are engaged in DNA repair, where they are activated by single and double DNA strand breaks. Their involvement in DNA repair appears to require the addition of PAR-chains (here referred to as PARylation) to the PARP enzyme itself or to other proteins involved in the repair process.
• PARylation the addition of PAR-chains
• AIMP2 and DX2 expressing vectors were introduced into HEK 293 cells. The cells were incubated for 24 hours, lysed, and immunoprecipitation was performed.
• PARylation is a post-translational process, regulating biological events such as DNA damage response and apoptosis. Szabo C et al. (1996) and Virag 1 et al. (1998).
• PARP-1 is an enzyme that recognizes damaged DNA in the nucleus, forms PAR chains, and induces degradation of damaged proteins and cell death through the PARylation.
• AIMP2 or DX2 affects PARP-1 activation (e.g., PARP-1 cleavage) and PARylation under oxidative stress conditions
• SH- SY5Y cells were transfected with vectors expressing empty vector (EV), AIMP2, or DX2 and then treated with hydrogen peroxide (H2O2) for 4hrs.
• AIMP2-transfected cells showed significantly increased cleavage of PARP-1 when compared to the expression seen in other transfected cells under oxidative stress conditions. However, PARP-1 cleavage was not observed in DX2-transfected cells (FIG. 7F). The PARylation of AIMP2 was increased in the presence of H2O2, but the PARlylation of DX2 was not altered (FIG. 7G).
• DX2 is insufficiently expressed, AIMP2 forms homodimers, interacts with PARP- 1 , activates PARP- 1 , and induces neuronal cell death.
• DX2 has a significantly higher binding affinity to PARP- 1 than AIMP2, the binding affinity between AIMP2 and PARP-1 was decreased in DX2 expressing cells, thereby leading to the inhibition of PARP-1 activity and the reduction in neuronal cell death.
• DX2 is a main regulatory protein in AIMP2-induced PARP-1 activation and cell death (FIG. 71).
• DX2 compromises the pro-apoptotic activity of AIMP2 via the competitive inhibition of AIMP2 binding to PARP- 1.
• Example 3 DX2 attenuates H2O2-induced neuronal cell death.
• DX2 is an inhibitory molecule of oxidative stress-induced PARP- 1 cleavage
• determination of whether DX2 suppresses cell death in the neuronal cells was further investigated.
• N2A cells Neuro-2a, mouse neuroblastoma cell line
• MTT analysis was performed to determine the level of cell death.
• DX2 expressing cells have significantly reduced cell death compared to control cells under oxidative stress conditions (FIG. 8C).
• FIG. 8C the anti-apoptotic effect of DX2 seems to be much stronger than apoptosis induction by AIMP2 in the co-existent condition of the two-proteins.
• TRAF dependent pathway DX2 compromises the TNF-a -dependent pro-apoptotic activity of AIMP2 via the competitive inhibition of AIMP2 binding to TRAF2.
• DX2 compromises the TNF-a -dependent pro-apoptotic activity of AIMP2 via the competitive inhibition of AIMP2 binding to TRAF2.
• Ocular inflammation is a common cause of visual impairment. Olivares-Gonzalez E et al. (2021). It is considered to be a chronic inflammation with sustained activation of glial cells (microglia, astrocytes) and recruitment of other immune cells into the nervous tissue (brain, spinal cord, or retina). Streit WJ (2004). Microglia cells, the resident innate immune cells in the retina and other nervous tissue, are potential cellular regulators of inflammation. Karlstetter M et al. (2015). Activated microglia and macrophages secrete pro-inflammatory mediators such as TNF- alpha and t on retinal cells (e.g., photoreceptor cells). Murakami Y et al. (2019). In the eye, TNF- a appears to participate in the pathogenesis of inflammatory, edematous, neovascular, and neurodegenerative diseases. Rodrigues EB et al. (2009).
• TRAF2 plays a crucial role in cell death.
• TRADD TNF Receptor Associated Death Domain
• TRAF2 binds to TNFR1, which is a TNF-a receptor and then, the activated complexes mediate IKB kinase (IKK) activation.
• IKK IKB kinase
• caspase-8 caspase-8 is inactivated
• TRAF2 is released from the TRADD complex, and the released TRAF2 is ubiquitinated by cIAP, which is a E3 ubiquitin ligase, promoting cell death.
• AIMP2 promotes the ubiquitination of TRAF2 by cIAP and stimulates cell death. Choi JW et al. (2009).
• HEK293 cells were transfected with NF-kB -luciferase vector and the cells were selected by G418 (Img/ml) for 1 week to establish the stable expressing cells. After the selection of cells survived, the cells were transfected with the pcDNA3.1 empty vector (as the control) or pcDNA 3.1 expression vector encoding the AIMP2 or DX2. Sixteen hours later, the transfected cells were treated with TNF-a (20 ng/ml) for 12 hours. The cells were harvested, and cell lysates were prepared for quantitation of luciferase using a luciferase assay kit following the manufacturer’s protocol (Promega).
• N2A and mouse primary neuron cells were transfected with EV (empty vector) or DX2 expression plasmid.
• the transfected cells were incubated in the presence or absence of TNF-a (20 ng/ml) + cycloheximide (CHX, 10 uM) for 6 h. Cell viability was then evaluated using a MTT assay. As shown in FIGS.
• DX2 overexpression did not affect cell viability of N2A or mouse primary neuron cells under normal conditions (con); however, upon TNF-a treatment, the cell death of DX2 transfected cells (DX2) were significantly reduced compared to the EV (empty vector) transfected cells.
• DX2 competes with AIMP2 and interacts with TRAF2 more strongly than AIMP2, thus inhibiting AIMP2-stimulated neuronal cell apoptosis in TNF-a-activated conditions.
• Example 5 Design of AAV2-DX2: DX2-coding self-complementary AAV2 [0202] As it is commonly difficult to treat retinal degenerative disease (RDD) using usual pharmacological methods, more effective strategies are being sought. Among these alternatives, gene therapy can be a strategy to access RDD and treat diseases, such as RP with minimal invasiveness of subretinal micro-injection.
• RDD retinal degenerative disease
• gene therapy can be a strategy to access RDD and treat diseases, such as RP with minimal invasiveness of subretinal micro-injection.
• AAV vectors are efficient shuttles that are able to deliver a transgene to the retinal cells, with their efficiently transducible character to non-dividing retinal cells.
• AAV is also appreciated as a suitable gene delivery approach because of its relative safety due to a lack of pathogenicity.
• AAV2 adeno-associated virus serotype 2
• SH-SY5Y cells were infected with 10, 100, 1000 or 104 MOI (Multiplicity of infection) of ssAAV-GFP (Green Fluorescent Protein) or scAAV-GFP. After 48 hours, GFP expression was measured by microscopy.
• FIG. HA represents a percentage of GFP positive cells and
• FIG. 11B shows a microscopy image of GFP expressing cell. In FIGS. HA and 11B, scAAV was more effective in virus infection rates when both viruses were used to treat SH- SY5Y at different concentrations.
• Example 6 AAV2-DX2 transduction suppresses neuronal death-associated cellular signaling.
• AAV2-DX2 compromises the H2O2 induced cytotoxic effects in the MEF (Mouse embryonic fibroblasts), hepatocytes, MSC (Mesenchymal stem cells) as well as in the primary neurons (Neuron), the cells were infected with 104 MOI of AAV-DX2 (AAV2- DX2) for 48 hours. Then, the transduced cells were treated 400 pM H2O2 for 4 hours to induce cell death and cell viability was measured by MTT analysis (FIG. 12A).
• AAV2-DX2-transduced cells commonly showed significant decreases in cell death when compared with AAV-GFP-transduced cells (GFP) after H2O2-treatment (FIG. 12A), implying that AAV2-DX2 is an effective anti-apoptotic agent.
• FFP AAV-GFP-transduced cells
• FIG. 12B cell viability was not affected by the presence or absence of AAV2-DX2 (FIG. 12B). Therefore, DX2 functions only when a stressor induces apoptotic conditions.
• RNA sequencing was conducted on neuronal cells after induction DX2 gene by an AAV delivery system.
• SK-N-SH neuroblastoma cells were infected with either scAAV2-GFP or AAV2-DX2. Forty-eight hours following infection, the cells were harvested, RNA was isolated and RNA sequencing was performed.
• DX2 was predicted to be involved in suppression of p53-associated cell death pathway and TNF-a or interleukin-related signaling (FIG. 12C).
• the gene set was analyzed based on two cell types, SK-N-SH (FIG. 12D) and N2A (FIG.
• DEGs differently expressed genes
• Example 7 DX2 increases 661 W cell viability, a retinal photoreceptor and ganglion precursorlike cell line.
• FIG. 13 shows that DX2 increased retinal photoreceptor neuronal cell viability. 661W cells were seeded at 5 x 10 A 4 cells per well in a 96-well plate, followed by infection with AAV-DX2 virus at an 5000MGI. After 48 hours, cells were treated with varying concentrations of H2O2. Cell viability was assessed 24 hours later using the MTT assay.
• AAV-DX2 661W cells were infected with AAV-DX2 then cell death was induced with H2O2 treatment, the mRNA levels of the pro-apoptotic marker BAX was assessed. The results showed that in cells infected with the control virus, AAV- GFP, BAX levels increased more than 2.5-fold upon H2O2 treatment compared to untreated cells. However, in cells treated with AAV-DX2, BAX levels were significantly reduced when treated with H2O2 compared to the AAV-GFP treated group.
• FIG. 14 shows that DX2 reduces the levels of BAX induced by H2O2.
• 661W cells were infected with either AAV-GFP or AAV-DX2 at an MOI of 5,000 each. After 72 hours, cells were treated with 2 mM H2O2 for 6 hours, and then the RNA levels of BAX were assessed.
• FIG. 16 shows that DX2 binds to KRS similarly to AIMP2.
• Cells transfected with 2 pg of Flag-tag- AIMP2 and Flag-tag-DX2 were subjected to cell lysis using NP-40 lysis buffer. Subsequently, a mixture of 15 pl agarose A/G plus beads and KRS antibody was used for the pulldown assay.
• AAV2-DX2 was evaluated in three RDD animal models in mouse and rabbit, i.e., laser-induced choroidal neovascularization, Mdml-/-(CRISPR/Cas9 KO) and Sodium iodate induced retinal degeneration model. Overall, AAV2-DX2 was determined to be effective in both the prevention and therapeutic settings.
• Example 9-1 AAV2-DX2 shows preventive effect of DX2 in the mouse Mdml-/-(CRISPR/Cas9 KO) model.
• Mdml Mouse double minute 1
• CC connecting cilium
• FIG. 18G shows integrity and proliferation of RPE (Retinal Pigment Epithelial). Transduction of DX2 gene resulted in the recovery of RPE integrity by activating proliferation of RPE.
• FIG. 18H shows PR (Photoreceptor) recovery. Transduction of DX2 gene resulted in recovery of PR population by activating proliferation of PR.
• FIG. 181 shows cellular proliferation of RPE and PR. Ki67 expression was measured to analyze proliferation in RPE and photoreceptor layers. Proliferation in RPE (left panel) and photoreceptor outer segment layer (right panel) was significantly higher in the AAV2-DX2 transfected sample.
• FIGS. 18J-18O show functional recovery of retina.
• AAV2-DX2 transduction showed increased a-wave amplitude (FIG. 18L) and reduced latency (FIG. 18M) compared to the RDD model (mdml-/-) or the negative control (mdml-/- + AAV-GFP), indicating that DX2 expression reduces damage of photoreceptors’ electrophysiological function and visual acuity.
• AAV2-DX2 transfected sample did not show a change in b-wave amplitude (FIG. 18N) but reduced the latency (FIG. 180) compared to the RDD model (mdml-/-), which suggests that DX2 affects only the RPE and photoreceptor, but not the bipolar cells (post-photoreceptors neurons).
• Electroretinography of AAV2-DX2 transduced sample showed increased regaining of normal ERG graph format (FIGS. 18J and 18K).
• AAV2-DX2 transfected sample showed slightly increased a-wave amplitude (FIG. 18L) and reduced latency (FIG. 18M) than the dry AMD (mdml-/-) indicating that the DX2 expression reduced the damage of photoreceptors’ electrophysiological function.
• the AAV2-DX2 transduced sample did not show a change b-wave amplitude (FIG. 18N) but reduced the latency (FIG. 180) than in the RDD model (mdml-/-).
• Mdml-/-(CRISPR/Cas9 KO) mouse was generated to present progressive photoreceptor and RPE degeneration.
• the animals were housed in individual cages under specific pathogen-free conditions and a constant environment condition (21°C - 23°C temperature, 50-60% humidity and 12-h light/dark cycle) in the animal facility.
• AAV2-DX2 and Negative control (AAV2-GFP) injection at Sub-retinal space at 3 weeks old. Histological measurements and functional recovery of retina were performed at 3 -months old.
• Three-week-old Mdml -/- mice were injected at subretinal space by trans-scleral injection to minimize retinal wound with AAV2-DX2/ AAV2-GFP in a volume of 4pl using a 38G sterile micro-tip needle (INCYTO, KR).
• AAV2-GFP/DX2 was injected into the same animal.
• AAV2- GFP was injected at OS (left).
• AAV2-DX2 was injected at OD (right).
• mice were induced to anesthesia with Avertin (1%) and anesthesia was maintained with 3% isoflurane inhalation and put on heating pad to maintain their physiological condition.
• a drop of 2% hypromellose solution drop was placed on the Rodent Contact Lens with Silver-embedded Thread Electrode to keep contact with the cornea and to keep it moistened.
• Mice were placed under the 76 mm diameter Ganzfeld dome for darkness and uniform illumination of the eyes. Measurements were performed under ISCEV-Extended full-field ERG standards protocols. The data were analyzed using ERGVIEW and the combined standard Rod&Cone response value was selected to analyze with a flash intensity of 3000mcd.s/m2, 0.10Hz.
• A-wave analysis was performed for photoreceptor cell function.
• B-wave analysis was performed for bipolar and horizontal cell function. Amplitude and latency values for a-wave and b-wave were analyzed.
• mice were euthanatized after ERG and their eyeballs were harvested. The eyeballs were fixed in 4% PFA overnight at 4°C. Eyeballs were dehydrated at 30% sucrose and embedded with the OCT compound for tissue cryosection. All retina cryosection samples were acquired from the optic nerve containing section with 10 pm thickness. Retina cryosections were analyzed. H&E was used for layer thickness analysis. Layer thickness analysis was performed with Leica LAS program. Immunofluorescence was used for RPE65 and Opsin expression, and Proliferation evaluation (Ki67). Immunofluorescence ROI set and overlapping coefficient measurements were measured with Image J.
• Example 10 AAV2-DX2 show preventive potency of DX2 in the Laser-induced choroidal neovascularization (CNV) mouse model.
• Example 10-1 AAV2-DX2 treated mice attenuates laser-induced choroidal neovascularization.
• Laser-induced choroidal neovascularization (CNV) model is a widely used animal model for retinal degenerative disease (RDD). In this model, laser is used to disrupt Bruch’s membrane, which allows the underlying choroidal vessels to penetrate and grow into the space underneath the pigment epithelium.
• Subretinal injection of scAAV2-GFP (control) or AAV2-DX2 to 5-wk-old male C57BL/6 mice (n 12) were conducted (Day 0). Twenty-one days after injection, CNV was produced by laser photocoagulation (Day 21).
• DX2 injected mice showed reduced CNV area compared to GFP injected mice in ICG angiography (FIG. 19A). Also, choroid flat mounts stained with isolectin-B4 demonstrates significant reduction in CNV formation areas in DX2 injected mice (FIG. 19A).
• the ratio of leaky area to CNV area were estimated by measuring the total hyperfluorescent area using fluorescein angiography (FA) and the CNV area using ICGA (FIG. 19B).
• Inflammatory cells, in particular (macrophages) have been histologically demonstrated near/within degenerative lesions, including areas of Bruch membrane breakdown, RPE atrophy, and CNV.
• VEGF vascular endothelial growth factor
• DX2 treated mice showed less VEGF expression compared to GFP treated mice. This data also showed preventive effect of DX2 on CNV model mouse.
• the animals were housed in individual cages under specific pathogen-free conditions and a constant environment condition (21°C - 23°C temperature, 50-60% humidity and 12-h light/dark cycle) in the animal facility.
• the mouse Ocular sinister (OS, left eye) in each group treated AAV- GFP and Ocular Dexter (OD, right eye) treated AAV-DX2. (Injection: 5x108 vg).
• OS left eye
• OD Ocular Dexter
• AAV-DX2 AAV-DX2.
• injection 5x108 vg
• RPE layer of eye fundus induce laser to make 3 weeks-, 3 months-retinal degenerative disease model, respectively.
• the mouse eyeball is isolated.
• AAV2-GFP was injected at OS (left).
• AAV2-DX2 was injected at OD (right). Retract the syringe slowly. Apply eye moisturizing drops to keep the eye hydrated. Continue to monitor the animal until it regains sternal recumbency.
• [0239] Mouse Laser- Induced CNV Model protocol.
• phenylephrine hydrochloride (2.5%) for dilation.
• administer another drop of tropicamide Once eye is sufficiently dilated, proceed to laser procedure. Adjust the placement of mouse on the mouse-stage, so that it is ideally positioned for visualization of optic nerve. Orient the mouse on its holder so it lies horizontally, perpendicular to slit lamp beam, with the head at one side and tail at the other.
• the coverslip acts as a contact lens to flatten the cornea. Look through slit lamp and with free hand toggle focus until retina can be visualized. The retina will have a light-yellow/red color depending on the location visualized, distinct, red vessels will be visible. Slowly and carefully manipulate mouse head and/or coverslip until visualizing the optic nerve. The optic nerve will be yellow in color with multiple vessels radiating from it.
• Proper focus is achieved by having the sharpest and clearest laser beam. If the aiming beam looks oval or out of focus, toggle slit lamp focus or re -position glass coverslip. Once the aiming beam is focused on RPE, initiate laser administration using the laser’s foot trigger. Watch for the appearance of a bubble immediately after laser administration. The outline of the laser shot should be clear and not hazy in any way. Repeat previous 3 steps for all desired CNV positions (usually at 3, 6, 9, and 12 o’clock positions around optic nerve). Record in a notebook the location and result of each laser shot administration and result (Successful, hazy, hemorrhage, etc.) of each administered shot for the eye. Be sure to place the laser in stand-by mode when not in use.
• the eyes were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) for 2 hours after removal of the cornea and lens. Posterior eyecups of the RPE/choroid/sclera were dissected, and the vitreous was removed. Eyecups were incubated overnight at 4°C with (AlexaFluor 647 or FITC)-conjugated Isolectin B4 (1:200, Invitrogen, Carlsbad, CA) to label invading choroidal vessels.
• paraformaldehyde Electrodehyde
• Example 11 AAV2-DX2 improves retinal degeneration in Sodium iodate (Sl)-induced rabbit RDD model.
• Sodium iodate (SI) is a widely used oxidant for generating retinal degeneration models by inducing the death of photoreceptor cells 40 41 and retinal pigment epithelial cells (RPE).
• Sodium iodate solution was injected once intravenously at the dose of 30 mg/kg to generate RDD model 42 .
• fundus images showed the RPE degeneration areas of sodium iodate induced group(G2) was significantly degenerated compared to normal control group (Gl).
• RPE degenerated area (%) levels of test articles treated groups (G5 and G6) were significantly recovered compared to G2 (FIGS. 20A-20B).
• Rhodopsin positive area (%) levels of G2 was significantly reduced compared to Gl (p ⁇ 0.001, p ⁇ 0.01). Rhodopsin positive area (%) levels of G5 and G6 were significantly recovered compared to G2 (p ⁇ 0.01) (FIGS. 20G-20J). Ganglion positive cells (%) levels of G2 was significantly reduced compared to Gl (p ⁇ 0.001). Ganglion positive cells (%) levels of G3, G4 and G6 were significantly recovered compared to G2 (p ⁇ 0.05).
• a wave and B wave amplitude levels of sodium iodate induced groups (G2) was significantly decreased compared to normal control group (Gl) (p ⁇ 0.001).
• FIGS. 21A-21B shows electroretinogram measurements of A wave (FIG. 21 A) and B wave (FIG 21B).
• sodium iodate (Sigma- Aldrich Co.) solution was injected once intravenously at the dose of 5 mg/kg to the animals, which were designated with degenerative eye diseases to be induced to, and at the date of 2nd induction (Day 14), sodium iodate solution was injected once intravenously at the dose of 30 mg/kg or 60 mg/kg in accordance with Settings for test group.
• retinal degeneration studies are conducted with 25- 30mg/kg sodium iodate solution.
• retinal degeneration was induced with very high dose of sodium iodate solution (60 mg/kg) to test whether DX2 is effective in neuronal survival, even in situations where the optic nerve is destroyed and total visual loss occurred.
• Test articles were injected once at the date of administration (Day 5). In case of intravitreal injection, the animals were anesthetized, and the test articles were injected intravitreally into the right eyeball with the syringe equipped 31gauge needle. In case of subretinal injection, the animal was anesthetized, and the test article were injected by trans-scleral route into the right eyeball with the syringe equipped 31 -gauge needle.
• Gl Normal control.
• G2 Vehicle control.
• I.Vt Intravitreal injection.
• S.R Subretinal injection, vg: viral genome.
• ERG electroretinogram
• CNV retinal degenerative disease
• EIG. 22B shows the analysis of CNV area using Isolectin B4 staining, the CNV area levels of all test article treated groups (G3, G4) and positive control (G5) values tends to decrease but did not reach statistical significance.
• Example. 13 AAV2-DX2 did not induce any histopathological abnormalities or neoplastic pattern in whole body organs.
• a whole-body histopathological analysis was performed to test the potential toxicity of any tumorgenicity of AAV2-DX2. As summarized in Table 4, AAV2-DX2 administered animals did not show any histopathological abnormalities or neoplastic pattern in the kidney, liver, spleen, brain, lymph node, lung etc. The survival was not affected compared to control (Gl) group.
• Example 14 Preliminary distribution study to confirm the injected tissue neuronal cell specific DX2 expression via AAV2-DX2-miR-3PT.
• Extracted organs Brain, Testis, Kidney, Liver, Lung, Spinal cord, Spleen, Stomach, Heart, Thymus, Pancreas, Lymph node and retina/choroid of right eyes (of both eyes of Gl).
• Extracted organs Brain, Testis, Kidney, Liver, Lung, Spinal cord, Spleen, Stomach, Heart, Thymus, Pancreas and Lymph node.
• Gl Normal control.
• G2 Vehicle control.
• I.Vt Intravitreal injection.
• S.R Subretinal injection, vg: viral genome.
• Retinal CNV area and efficacy evaluation were performed using retinal fluorescent fundus photography.
• the Image analysis was performed using ImageJ software (NIH, Bethesda, MD) to verify the fluorescence intensity of the irradiation site.
• Fluorescein intensity (%) (Fluorescein intensity value - average of background value(Gl) 1) )/average of fluorescein intensity value of G2
• RNA was isolated from rabbit ocular tissues and each organs using the RNA isolation Geneall hybrid-R Kit (GeneAll Biotechnology Co., Ltd., Korea) and reverse transcribed with the cDNA synthesis Kit (Toyobo, Japan).
• the complementary DNAs were then used for real time PCR using the qPCRBIO Sy Green Blue Mix (PCR Biosystems Ltd., UK). Amplification, detection, and data analysis were performed with a CFX96 touch (BioRad, USA). RT-PCR analysis of target gene using extracted organs and ocular tissues were carried out to confirm the quantity of target gene expression compared to GAPDH expression.
• Deng WT ea Correction to: Stability and Safety of an AAV Vector for Treating RPGR- ORF15X-Linked Retinitis Pigmentosa by Deng, W.-T. et al. Hum Gene Ther 2015;26(9):593- 602. DOI: 10.1089/hum.2015.035. Hum Gene Ther. Feb 2021;32(3-4):232-233. doi : 10.1089/hum.2015.035.correx.
• Pawlyk BS Bulgakov OV, Sun X, et al. Photoreceptor rescue by an abbreviated human RPGR gene in a murine model of X-linked retinitis pigmentosa. Gene Ther. Feb 2016;23(2):196- 204. doi: 10.1038/gt.2015.93. 10. Sun X, Park JH, Gumerson J, et al. Loss of RPGR glutamylation underlies the pathogenic mechanism of retinal dystrophy caused by TTLL5 mutations. Proc Natl Acad Sci U SA. May 24 2016;113(21):E2925-34. doi: 10.1073/pnas.1523201113.

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