Use of human nd6 nucleic acid in the preparation of drugs for preventing and treating mitochondrial retinal and optic neuropathy

Abstract

The application discloses an optimized nucleic acid for ectopic expression of human mitochondrial ND6 protein, a recombinant expression cassette, a recombinant adeno-associated virus and medical uses thereof. The optimized nucleic acid encodes human wild-type ND6 protein using nucleotide codons and is operably linked to a mitochondrial targeting sequence, so that the ND6 protein formed by transcription in the nucleus and translation in the cytoplasm can enter the mitochondria and participate in the assembly of respiratory chain complex I. The recombinant AAV2 containing the expression cassette can improve the retinal and optic nerve functional defects of a homogenous mt-ND6 mutant mouse in the pre-symptomatic or early symptomatic stage, improve the complex I activity, and improve the visual function, retinal ganglion cells and optic nerve structure after intravitreal administration. The treatment response in the late atrophy stage is lower than that in the early stage, indicating that the application is particularly suitable for early intervention of MT-ND6 mutation related retinal and optic nerve diseases.

Description

Technical Field

[0001] This invention belongs to the fields of gene therapy for mitochondrial diseases and ophthalmic biological agents, specifically involving the application of human ND6 nucleic acid in the preparation of drugs for the prevention and treatment of mitochondrial retinal and optic nerve diseases. It is a codon-optimized human ND6 nucleic acid, a mitochondrial targeted expression cassette containing the nucleic acid, recombinant adeno-associated virus particles, a pharmaceutical composition, and its application in the prevention, delay, or treatment of MT-ND6 mutation-related retinal and optic nerve diseases. Background Technology

[0002] Mitochondrial respiratory chain complex I, composed of multiple subunits encoded by both nuclear and mitochondrial genes, is a crucial protein complex for maintaining mitochondrial oxidative phosphorylation and cellular energy metabolism. ND6 is one of the subunits of complex I encoded by mitochondrial DNA. Mutations in the MT-ND6 gene can lead to abnormalities in the structure or function of the ND6 protein, thereby affecting the assembly and enzyme activity of complex I, resulting in impaired oxidative phosphorylation, reduced ATP production, and imbalances in reactive oxygen species homeostasis.

[0003] Retinal ganglion cells and their axons have high energy requirements and are particularly sensitive to mitochondrial dysfunction. Therefore, pathogenic mutations in MT-ND6 can cause abnormalities in the retina, optic nerve, and visual function, such as Leber hereditary optic neuropathy (LHON). LHON primarily manifests as painless, subacute, or acute vision loss, often affecting both eyes, and may be accompanied by peripapillary microvascular abnormalities, swelling of the retinal neurofibrillary layer, and later optic nerve atrophy. This disease is more common in young men, and some patients may experience severe and persistent central visual impairment. Currently, clinical treatment options for LHON remain limited, and treatment responses vary significantly among patients. There is still a lack of treatments that can stably correct mitochondrial gene defects and restore retinal and optic nerve function.

[0004] Gene therapy based on compensating for the function of normal genes or proteins is a potential strategy for treating MT-ND6 mutation-related diseases. However, due to the differences between the mitochondrial and nuclear genetic codes, the native mitochondrial ND6 gene sequence is difficult to translate correctly directly in nuclear expression systems. Ectopic expression strategies typically redesign the ND6 gene to use a nucleic acid sequence encoded by the nuclear genetic code, enabling it to be transcribed in the nucleus and translated in the cytoplasm. Simultaneously, guided by mitochondrial-targeting peptides, the expressed ND6 protein is introduced into the mitochondria to compensate for the functional defects of the endogenous mutant ND6 protein.

[0005] While existing technologies include concepts for ectopic expression of ND6, ND6 is a highly hydrophobic mitochondrial inner membrane protein. Its nucleic acid sequence design, codon optimization, mitochondrial targeting sequence, and expression cassette structure can all affect the expression efficiency, stability, mitochondrial delivery, inner membrane localization, and complex I assembly of the ND6 protein. These factors may further influence the therapeutic efficacy and safety of viral vectors when used intraocularly.

[0006] Therefore, it is still necessary to develop an optimized human ND6 nucleic acid and viral delivery system that can effectively express, accurately target mitochondria and participate in complex I assembly in retinal cells, in order to improve retinal and optic nerve functional defects caused by MT-ND6 mutations, and further clarify its applicable disease stages and therapeutic window. Summary of the Invention

[0007] The present invention aims to provide the application of human ND6 nucleic acid in the preparation of drugs for the prevention and treatment of mitochondrial retinal and optic nerve diseases. The nucleotide sequence of the human ND6 nucleic acid is shown in SEQ ID NO:1, and the amino acid sequence of the ND6 protein it encodes is shown in SEQ ID NO:2. The nucleic acid was redesigned according to the nuclear genetic code without altering the amino acid sequence of the human ND6 protein, and codon optimization was performed for transcription and cytoplasmic translation in the nuclear expression system.

[0008] The mitochondrial retinal and optic nerve diseases are associated with MT-ND6 mutations, including Leber hereditary optic neuropathy and other mitochondrial retinal or optic nerve diseases caused by MT-ND6 dysfunction.

[0009] After the human ND6 nucleic acid is expressed, the resulting ND6 protein enters the mitochondria under the guidance of the mitochondrial targeting sequence and participates in the assembly of mitochondrial respiratory chain complex I, thereby compensating for the ND6 functional deficiency caused by MT-ND6 mutation.

[0010] The expression of the human ND6 nucleic acid can enhance the activity of mitochondrial respiratory chain complex I, improve retinal and optic nerve functional and structural abnormalities caused by MT-ND6 mutations, including improving electroretinography, visual evoked potentials and visual behavior, and reducing damage to retinal ganglion cells and optic nerve axons.

[0011] The optimized human ND6 nucleic acid can be contained in a recombinant expression cassette and further constructed into a recombinant adeno-associated virus vector genome and a recombinant adeno-associated virus delivery system to achieve the expression of human ND6 protein in eye tissue and mitochondrial targeting and assembly of mitochondrial respiratory chain complex I.

[0012] This invention provides a recombinant expression cassette, comprising a promoter, a nucleic acid sequence encoding a mitochondrial targeting peptide, and the optimized human ND6 nucleic acid. Preferably, the recombinant expression cassette further comprises one or more of the following: a CMV enhancer, a Kozak sequence, WPRE, and SV40polyA. The nucleotide sequence of the recombinant expression cassette is shown in SEQ ID NO:4.

[0013] Preferably, the mitochondrial targeting sequence is located within the same open reading frame as the optimized human ND6 nucleic acid, enabling the expressed human ND6 protein to be introduced into the mitochondria. The mitochondrial targeting sequence is shown in SEQ ID NO:3.

[0014] The present invention provides a recombinant adeno-associated virus vector genome, wherein the vector genome includes the aforementioned recombinant expression cassette located between two adeno-associated virus inverted terminal repeat sequences.

[0015] The present invention also provides a recombinant adeno-associated virus (AAV) particle, the recombinant AAV particle comprising an AAV capsid and the aforementioned recombinant AAV vector genome packaged within the capsid. In other words, the recombinant AAV vector genome is a nucleic acid component packaged inside the viral capsid, and the recombinant AAV particle consists of the viral capsid and the recombinant AAV vector genome packaged within it. Preferably, the AAV capsid is an AAV2 capsid.

[0016] The present invention provides a pharmaceutical composition comprising the above-described recombinant adeno-associated virus particles and a pharmaceutically acceptable carrier or excipient.

[0017] This invention uses C57BL / 6J mice with a homogeneous mitochondrial ND6 P25L mutation to evaluate the therapeutic effects of the aforementioned recombinant adeno-associated virus particles or drug compositions on MT-ND6 mutation-related retinal and optic neuropathy. The mouse model can be used to detect changes in visual function, retinal structure, retinal ganglion cells, optic nerve axons, and mitochondrial respiratory chain complex I function after drug administration.

[0018] Compared with the prior art, the present invention has at least the following beneficial effects: 1. The optimized human ND6 nucleic acid described in this invention can be effectively expressed in mammalian cells. After intraocular delivery mediated by recombinant AAV2, exogenous ND6 can be continuously expressed in the retina, and related signals can be detected in optic nerve tissue; the expressed ND6 protein can enter the mitochondria under the guidance of a mitochondrial targeting sequence; 2. The expressed exogenous ND6 protein can participate in the assembly of retinal mitochondrial respiratory chain complex I and enhance the activity of complex I, thereby improving mitochondrial dysfunction caused by MT-ND6 mutation; 3. Intravitreal administration of recombinant AAV2-hoptND6 can improve visual dysfunction in ND6 mutant mice, including visual evoked potentials and visual behavior indicators. 4. Recombinant AAV2-hoptND6 can alleviate retinal ganglion cell damage in ND6 mutant mice and improve optic nerve axonal and related tissue structural abnormalities; 5. In wild-type mice, low, medium and high doses of recombinant AAV2-hoptND6 administered intravitreally demonstrated acceptable ocular safety within the observed time and dose ranges. 6. Treatment with recombinant AAV2-hoptND6 before the onset of symptoms or in the early stages of the disease is more effective than late administration after significant retinal and optic nerve atrophy has already occurred, indicating that the present invention is particularly suitable for early intervention in MT-ND6 mutation-related retinal and optic nerve diseases. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the genome structure and expression cassette of the AAV2-hoptND6 vector.

[0020] Figure 2 The results show the expression of hoptND6 in cells compared with other different versions of hND6.

[0021] Figure 3 The results show the expression of AAV2-hoptND6 in the retina at different time points after injection for each dose.

[0022] Figure 4 This describes the localization of exogenous ND6 in retinal mitochondria.

[0023] Figure 5 The results of exogenous ND6 participating in the assembly of complex I are shown in BN-PAGE.

[0024] Figure 6 The results show the localization of AAV2-hoptND6 in the retina and optic nerve.

[0025] Figure 7 The results represent the retinal safety of low, medium, and high doses of AAV2-hoptND6 in wild-type mice.

[0026] Figure 8 The retinal disease phenotypes of ND6 mutant mice at different ages.

[0027] Figure 9 This represents the improvement in retinal mitochondrial complex I function resulting from early AAV2-hoptND6 treatment.

[0028] Figure 10This is the result of early AAV2-hoptND6 treatment improving the number of retinal neurosensory cells (RGCs).

[0029] Figure 11 This represents the improvement in optic nerve density resulting from early AAV2-hoptND6 treatment.

[0030] Figure 12 This is the result of early AAV2-hoptND6 treatment improving visual evoked potentials (VEP).

[0031] Figure 13 This represents the improvement in visual behavioral response (qOMR) resulting from early AAV2-hoptND6 treatment.

[0032] Figure 14 The results of treatment with AAV2-hoptND6 in the atrophic stage. Detailed Implementation

[0033] The present invention will be further described in conjunction with the accompanying drawings and embodiments, but these embodiments do not constitute a limitation on the scope of protection of the present invention. Experimental materials, detection methods, and statistical methods not specifically described can be implemented according to conventional methods in the art.

[0034] Example 1: Optimization of the construction of human ND6 nucleic acid, recombinant expression cassette and recombinant adeno-associated virus.

[0035] Based on the amino acid sequence of the human mitochondrial ND6 protein, its coding sequence was redesigned using the nuclear genetic code. Codon usage frequency, GC content, potential aberrant splicing sites, repetitive sequences, and sequences that might affect RNA stability were optimized to obtain the optimized human ND6 nucleotide sequence shown in SEQ ID NO:1. This optimized human ND6 nucleotide sequence encodes the human ND6 protein shown in SEQ ID NO:2. The mitochondrial targeting sequence shown in SEQ ID NO:3 was placed at the 5′ end of the optimized human ND6 nucleotide sequence, and both were positioned within the same open reading frame to obtain the MTS-ND6 fusion nucleic acid sequence. This fusion nucleic acid sequence was placed under the regulation of the CMV promoter, and WPRE and SV40 polyA were sequentially linked to its 3′ end to obtain the complete ND6 recombinant expression cassette. The nucleotide sequence of the recombinant expression cassette is shown in SEQ ID NO:4.

[0036] To detect the expression and localization of exogenous hoptND6 protein, a FLAG-labeled hoptND6 tracer expression cassette was further constructed. The nucleic acid sequence encoding the FLAG epitope tag was ligated to the 3′ end of the hoptND6 coding sequence, and the MTS, hoptND6, and FLAG tags were positioned within the same open reading frame, resulting in the MTS-hoptND6-FLAG fusion nucleic acid sequence. The FLAG nucleotide sequence is shown in SEQ ID NO:5. The MTS-hoptND6-FLAG fusion nucleic acid sequence was then placed under the regulation of the CMV promoter, and WPRE and SV40 polyA were sequentially ligated to its 3′ end, resulting in a FLAG-labeled hoptND6 recombinant expression cassette. The nucleotide sequence of the FLAG-labeled recombinant expression cassette is shown in SEQ ID NO:6.

[0037] Both the tagless hoptND6 recombinant expression cassette and the FLAG-labeled hoptND6 recombinant expression cassette were prepared using a whole-genome synthesis method and synthesized by Beijing Qingke Biotechnology Co., Ltd. The synthesized recombinant expression cassettes were cloned into AAV vectors to obtain tagless hoptND6 recombinant AAV vector plasmids and FLAG-labeled hoptND6 recombinant AAV vector plasmids. The tagless hoptND6 recombinant AAV vector plasmid was named pAAV-hoptND6; the FLAG-labeled hoptND6 recombinant AAV vector plasmid was named pAAV-hoptND6-FLAG. Schematic diagrams of the two recombinant vectors are shown below. Figure 1 As shown, recombinant adeno-associated virus (AAV) was packaged using pAAV-hoptND6 and pAAV-hoptND6-FLAG plasmids, respectively, to obtain recombinant AAV particles containing an AAV2 capsid, named rAAV2-hoptND6 and rAAV2-hoptND6-FLAG, respectively. The packaging, purification, and preparation of the recombinant AAV were performed by Guangzhou Paizhen Biotechnology Co., Ltd.

[0038] Among them, rAAV2-hoptND6 is the main candidate virus for evaluating safety and therapeutic efficacy; rAAV2-hoptND6-FLAG is mainly used to detect the expression of exogenous ND6 protein, mitochondrial localization and its binding or assembly with mitochondrial respiratory chain complex I.

[0039] Example 2: Expression and localization detection of hoptND6.

[0040] 2.1 Comparison of cellular expression of different versions of human ND6 nucleic acid To compare the expression capabilities of the optimized human ND6 nucleic acid of this invention with other versions of human ND6 nucleic acid, expression vectors containing hoptND6 and other different versions of human ND6 coding sequences (hND6-v1, hND6-v2) were constructed. These expression vectors were introduced into 293T cells, with untransfected and / or empty vector transfected groups serving as controls. After transfection and culturing for 48 h, cells were collected and total protein was extracted. Western blotting was used to detect the expression levels of different versions of human ND6 protein. Results are as follows: Figure 2 As shown, under the same experimental conditions, the mRNA of hoptND6 described in this invention is significantly higher than that of other versions of human ND6 nucleic acid, indicating that the nucleotide sequence optimization of this invention is beneficial to the ectopic expression of human ND6 in the cytoplasm.

[0041] 2.2 Expression of different doses of rAAV2-hoptND6-FLAG in mouse retina To evaluate the retinal expression level and duration of rAAV2-hoptND6-FLAG after intraocular administration, mice were randomly divided into a control group, a low-dose group, a medium-dose group, and a high-dose group. The low, medium, and high doses were 5 × 10⁻⁶. 7 vg / eyeball, 5×10 8 vg / eyeball and 5×10 9 vg / eyeball. rAAV2-hoptND6-FLAG was injected intravitreally into the eyes of mice, while the control group received saline-buffered saline. Retinal tissue was collected at 1, 3, and 6 months post-injection. Total retinal protein was extracted, and the expression of exogenous ND6 protein was detected by Western blotting. Results are as follows: Figure 3 As shown, rAAV2-hoptND6-FLAG can be expressed in the mouse retina after intravitreal injection. Within the dose range tested in this example, the expression level of exogenous hoptND6 generally increased with increasing viral dose and could be persistently detected at multiple time points after injection. These results indicate that rAAV2-hoptND6 can mediate persistent exogenous human ND6 expression in retinal tissue.

[0042] 2.3 Localization detection of exogenous ND6 in mitochondria To determine whether exogenously expressed ND6 protein could be introduced into retinal mitochondria, retinal tissue was collected 3 months after rAAV2-hoptND6-FLAG injection, and mitochondrial and cytoplasmic components were separated using differential centrifugation. Proteins from each component were extracted and analyzed by Western blotting. Exogenous ND6 was detected using a FLAG-tagged antibody, SDHA was used as a marker for mitochondrial components, and GAPDH was used as a marker for cytoplasmic components. Results are as follows: Figure 4As shown, exogenous ND6 signals were mainly detected in the mitochondrial components of the retina. The detection results of mitochondrial and cytoplasmic markers indicated that the obtained subcellular components had good separation efficiency. These results demonstrate that, guided by MTS, the exogenous human ND6 protein encoded by hoptND6 can enter the mitochondria of retinal cells.

[0043] 2.4 Exogenous ND6 participates in the assembly of mitochondrial respiratory chain complex I. To test whether exogenous ND6 can participate in the assembly of mitochondrial respiratory chain complex I, mitochondria were isolated from the retinal tissue of mice in the rAAV2-hoptND6-FLAG injection group and the control group, and mitochondrial membrane proteins were dissolved using a mild detergent.

[0044] The obtained mitochondrial proteins were subjected to BNG-PAGE electrophoresis, followed by Western blotting. Exogenous ND6 was detected using a FLAG-tagged antibody, with SDHC-labeled complex II used as a control. Results are as follows: Figure 5 As shown, exogenous ND6-related signals were detected in complex I and respiratory chain supercomplex in the injection group, while no signals were detected in the control group. This indicates that the exogenous human ND6 expressed by rAAV2-hoptND6-FLAG can not only enter retinal mitochondria, but also participate in the assembly of mitochondrial complex I.

[0045] 2.5 Distribution of exogenous ND6 in the retina and optic nerve To evaluate the intraocular tissue distribution of rAAV2-hoptND6-FLAG after intravitreal administration, retinal and optic nerve tissues were collected 3 months post-injection. Immunofluorescence staining was used to detect the distribution of rAAV2-hoptND6 in the retina and optic nerve. Results are as follows: Figure 6 As shown, in the high-dose injection group, exogenous ND6 was widely distributed in the retina, with a more pronounced distribution in the retinal ganglion cell layer, and varying degrees of signal could be detected in other layers of the retina. Exogenous ND6 protein-related signals were also detected in the optic nerve tissue.

[0046] Example 3: Safety evaluation of different dosages.

[0047] To evaluate the safety of intraocular administration of rAAV2-hoptND6-FLAG, mice were randomly divided into a control group, a low-dose group, a medium-dose group, and a high-dose group. The low, medium, and high doses were 5 × 10⁻⁶. 7 vg / eyeball, 5×10 8 vg / eyeball and 5×10 9 vg / eyeball. Retinal tissue was collected at 2 weeks, 4 weeks, 2 months, and 6 months post-injection, and ocular safety was evaluated using HE histological parameters. Results are as follows: Figure 7The results show that, within the dose range and observation period of this embodiment, AAV2-ND6 did not cause unacceptable damage to retinal structure or function.

[0048] Example 4: Treatment response of mice in the early treatment group.

[0049] First, the ND6 retinal phenotypes of different ages were detected by OCT, and the results are as follows: Figure 8 As shown, ND6 mutant mice were observed to develop the disease at 3 months of age.

[0050] 4.1 Experimental Grouping: Wild-type mice with C57BL / 6J background and mitochondrial ND6 P25L mutant mice were selected. Wild-type mice and ND6 mutant mice were divided into four groups: wild-type control mice, wild-type mice that received rAAV2-hoptND6 injection, ND6 mutant mice that did not receive injection, and ND6 mutant mice that received rAAV2-hoptND6 injection.

[0051] 4.2 rAAV2-hoptND6 injection therapy: RAAV2-hoptND6 was administered to mice at 2 months of age via intravitreal injection. The dosage was 5 × 10⁻⁶. 9 vg / eye, administration volume 2 μL. All mice were kept under a 12:12 hour light / dark cycle with free access to food and water, and were observed for up to 4 months post-administration.

[0052] The study followed ethical guidelines and animal experimentation norms, and obtained the relevant experimental permits and approvals.

[0053] 4.3 Treatment outcome assessment: (1) Functional detection of retinal mitochondrial complex I Figure 9 The results of histochemical staining were used to detect the activity of mitochondrial complex I in the mouse retina. The results showed that the activity of mitochondrial complex I in the retina of mutant mice was significantly increased after treatment with rAAV2-hoptND6.

[0054] (2) Morphological examination of the retina and optic nerve Figure 10 The image shows the results of detecting the number of Brn3a-positive ganglion cells in the mouse retina using retinal immunofluorescence technology. Figure 11 Transmission electron microscopy was used to analyze the axonal density of the optic nerve in mice. The results showed that the number of Brn3a-positive ganglion cells and the axonal density of the optic nerve were significantly increased in the retina of mutant mice treated with rAAV2-hoptND6.

[0055] (3) Visual function testing in mice Figure 12 , Figure 13 The figures show the results of detecting visual evoked potentials (VEP) and visual behavioral responses (qMOR) in mice. The results show that the VEP amplitude and qMOR of mutant mice in the rAAV2-hoptND6 treatment group were significantly restored, indicating the recovery of visual function in the mice.

[0056] Example 5: Treatment response of mice in the atrophy phase treatment group.

[0057] This invention also involves intravitreal injection of AAV2-ND6 into 12-month-old ND6 mutant mice that have developed significant retinal and optic nerve atrophy, and evaluation of the treatment effect is performed 3 months after administration. Figure 14 The results showed that, within the dosage and observation period used in this embodiment, the number of retinal Brn3a-positive ganglion cells in the 12-month-old treatment group did not significantly improve. This suggests that when significant irreversible loss of retinal ganglion cells and optic nerve axons has occurred, simply restoring ND6 function may not be sufficient to produce significant tissue and visual function recovery.

[0058] Therefore, the preferred application stage of this invention is before the onset of symptoms or in the early stage of the disease, i.e., when sufficient retinal ganglion cells and optic nerve axons are still preserved. This comparative example is not intended to negate the product use of the optimized nucleic acid, recombinant expression cassette, or AAV2 viral particles themselves, but rather to illustrate the impact of disease stage on treatment response.

Claims

1. The application of a human ND6 nucleic acid in the preparation of drugs for the prevention and treatment of mitochondrial retinal and optic nerve diseases, characterized in that, The nucleotide sequence of the human ND6 nucleic acid is shown in SEQ ID NO:1, and the amino acid sequence of the ND6 protein it encodes is shown in SEQ ID NO:

2.

2. The application according to claim 1, characterized in that, The mitochondrial retinal and optic nerve diseases are associated with MT-ND6 mutations, including Leber hereditary optic neuropathy and other mitochondrial retinal or optic nerve diseases caused by MT-ND6 dysfunction.

3. The application according to claim 1, characterized in that, The human ND6 nucleic acid-expressed ND6 protein enters the mitochondria via a mitochondrial targeting sequence and participates in the assembly of mitochondrial respiratory chain complex I.

4. The application according to claim 1, characterized in that, The drug enhances the activity of mitochondrial respiratory chain complex I and improves the functional and structural abnormalities of retinal ganglion cells and optic nerve caused by MT-ND6 mutations.

5. The application according to claim 1, characterized in that, The human ND6 nucleic acid is contained in a recombinant expression cassette, which includes a CMV promoter, a nucleic acid sequence encoding a mitochondrial targeting peptide, the human ND6 nucleic acid, WPRE, and an SV40 polyadenylation signal sequence; the recombinant expression cassette is packaged in an adeno-associated virus capsid to form recombinant adeno-associated virus particles.

6. The application according to claim 5, characterized in that, The mitochondrial targeting sequence is shown in SEQ ID NO:3, and the nucleotide sequence of the recombinant expression cassette is shown in SEQ ID NO:4.

Images