CN117899199A - Medicine for treating photoreceptor cell degeneration - Google Patents

Abstract

The present disclosure relates to a medicament for treating photoreceptor cell degeneration. The present disclosure discloses the use of neuropeptides Y (NPY) or compositions containing NPY, nucleic acids encoding NPY, nucleic acid constructs or vector systems comprising NPY-encoding nucleic acid, viruses comprising NPY-encoding nucleic acid, or cells comprising NPY-encoding nucleic acid in the manufacture of a medicament for treating photoreceptor cell degeneration, such as retinitis pigmentosa, in a subject. The present disclosure innovatively uses AAV as a vector, which can be stably expressed in vivo for a long period of time.

Description

Medicine for treating photoreceptor cell degeneration

Technical Field

The disclosure belongs to the biomedical field, and in particular relates to a pharmaceutical composition for treating photoreceptor cell degeneration by using rAAV as a carrier based on NPY gene therapy and application thereof.

Background

The retina is a photosensitive membrane located in the back of the eye, with special organoleptic properties, capable of capturing light by photoreceptor cells (photoreceptors, photoreceptor cell) which are separated into cone cells (cone cells) and rod cells (rod cells) according to their ability to be more active in bright or dim light, respectively, at which level they are converted into electrical signals by the light transduction process and the electrical impulses are further integrated and processed into neurons, forming an image in the central nervous system (Central Nervous System, CNS).

Retinitis pigmentosa (RETINITIS PIGMENTOSA, RP) is a chronic, progressive, hereditary, dystrophic retinopathy, also known as retinitis pigmentosa. The main characteristics are that the functions of retinal photoreceptors and pigment epithelium are damaged progressively, the pathology changes into progressive degenerative diseases starting from the equatorial rod cells, with different degrees of atrophy of each retinal layer, glial and vascular occlusion sclerosis, pigment epithelium cytochrome loss and migration into the retina, and finally, the irreversible and progressive death of the retinal photoreceptor cells are caused. Its main clinical features are progressive loss of vision, night blindness and obvious pigmentation.

The incidence rate of the retinitis pigmentosa is about 1/5000-1/3000, and in China, the incidence rate of RP is also increased year by year, about 1/1000. The disease usually occurs in early childhood or adolescence, aggravates in adolescence, gradually reduces vision, is serious to blindness, is usually a binocular disease, is extremely rare in monocular disease, and is one of important causes of blindness. Currently, there is no drug or method that can completely cure RP, and most of the existing treatment schemes are symptomatic treatment and slowing down disease progression to achieve the goal of improving vision. There are many reports on primary treatment of RP including drugs, lasers, surgical treatments, gene therapies, cell therapies, and the like. However, most of the conventional therapeutic methods have ambiguous curative effects.

Retinal dystrophy (Retinal Dystrophies, also known as hereditary retinal degeneration) is a large group of hereditary eye diseases, resulting in irreversible vision loss and blindness, due to the death of retinal photoreceptor cells caused by mutations in one or more genes. A variety of therapeutic regimens for the treatment of retinal dystrophies are currently being actively sought.

Cone rod dystrophy (Cone-rod Dystrophy) is one of the inherited macular degeneration diseases, mainly damaging Cone cells, with varying degrees of rod cell damage. This disease mainly involves the macular area, and retinal pigment degeneration in the peripheral part may also occur later. Cone cell damage occurs earlier, so the main symptom is vision loss, acquired color vision abnormality, night blindness when rod cells are damaged. Therefore, also called central retinal pigment degeneration. At present, although various studies have good results in terms of therapeutic intervention of hereditary retinal diseases, there is no clear therapeutic approach.

Adeno-Associated Virus (AAV) is a very fast and popular important tool for gene delivery due to its high safety, wide host range, low immunogenicity and long-term efficient expression of foreign genes. To date, seven global gene therapy drugs using recombinant AAV as a delivery tool are marketed in batches, glybera from Zolgensma, uniqure, luxtura from Spark Therapeutics, hemgenix from CSL behring, roctavian from Elevidys, biomarin and Upstaza from PTC therapeutics from Sarepta Therapeutics, respectively, from nova (novartis), and several hundred gene therapy drugs using recombinant AAV as a delivery tool have entered clinical trials. Luxturna, approved by the FDA in 2017, is the first RP gene therapy drug, and gene therapy is one of the most potential therapeutic approaches for the current treatment of retinal diseases, especially photoreceptor degeneration.

Disclosure of Invention

The present disclosure provides the use of neuropeptide Y (NPY) or a pharmaceutical composition comprising neuropeptide Y (NPY) in the manufacture of a medicament for treating photoreceptor cell degeneration such as Retinitis Pigmentosa (RP), retinal dystrophy or degeneration, rod cone dystrophy, and the like in a subject.

The present disclosure provides the use of a nucleic acid encoding neuropeptide Y (NPY), a construct or vector system comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY), or a pharmaceutical composition comprising the same, in the manufacture of a medicament for treating photoreceptor degeneration such as Retinitis Pigmentosa (RP), retinal dystrophy or degeneration, rod cone dystrophy, or the like in a subject.

In another aspect, the present disclosure provides a method of treating photoreceptor cell degeneration, such as Retinitis Pigmentosa (RP), retinal dystrophy or degeneration, rod cone dystrophy, or the like, in a subject comprising administering to the subject a therapeutically effective amount of neuropeptide Y (NPY) or a composition comprising neuropeptide Y (NPY), a nucleic acid encoding neuropeptide Y (NPY), a construct comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY).

In another aspect, the present disclosure provides the use of neuropeptide Y (NPY) or a composition containing neuropeptide Y (NPY), a nucleic acid encoding neuropeptide Y (NPY), a construct comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY) in a method of treating photoreceptor cell degeneration such as Retinitis Pigmentosa (RP), retinal dystrophy or degeneration, rod cone dystrophy, or the like in a subject.

In some embodiments, the neuropeptide Y in the present disclosure is a mammalian-derived NPY or variant thereof, preferably a human neuropeptide Y (hNPY), a human neuropeptide Y mature (hmNPY), a rat-derived NPY (rNPY), a mouse-derived NPY (musNPY), or variant thereof.

In some embodiments, the hNPY comprises an amino acid sequence selected from SEQ ID No. 1, having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 1.

In some embodiments, the nucleic acid encoding an hNPY comprises a nucleic acid sequence selected from SEQ ID No.2 having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 2.

In some embodiments, the hmNPY comprises an amino acid sequence selected from SEQ ID NO. 3, having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 3.

In some embodiments, the nucleic acid encoding hmNPY comprises a nucleic acid sequence selected from SEQ ID NO.4, having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 4.

In some embodiments, the rNPY comprises an amino acid sequence selected from SEQ ID NO. 5, having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 5.

In some embodiments, the nucleic acid encoding rNPY comprises a nucleic acid sequence selected from SEQ ID NO. 6, having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 6.

In some embodiments, the musNPY comprises an amino acid sequence selected from SEQ ID NO. 7, having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 7.

In some embodiments, the nucleic acid encoding musNPY comprises a nucleic acid sequence selected from SEQ ID NO. 8, having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 8.

In some embodiments, the vector comprising the encoded NPY further comprises at least one of the following functional elements: inverted Terminal Repeat Sequences (ITRs), promoters, regulatory elements and polyA sequences.

In some embodiments, the vector comprising the encoded NPY is selected from at least one of an AAV vector, an adenovirus vector, a lentiviral vector, an RNA viral vector, or a vaccinia viral vector.

In some embodiments, the virus comprising a nucleic acid encoding NPY is an AAV virus, preferably wherein the serotype of an AAV capsid protein in the AAV virus is selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8R, AAV, AAV10, AAVrh10, or AAVDJ.

In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

In some embodiments, a second agent that can treat photoreceptor cell degeneration, such as retinal pigment degeneration (RP), retinal dystrophy or degeneration, rod cone dystrophy, and the like, is further included in the composition.

In some embodiments, the subject is a mammalian subject, preferably a human subject.

In some embodiments, the medicament is suitable for intraocular, intravenous, intra-arterial, subcutaneous, intramuscular, intratracheal, or inhalation administration.

Advantageous effects

The present disclosure innovatively uses neuropeptide Y (NPY) -based gene therapy for the treatment of photoreceptor degeneration. The rAAV is innovatively used as a vector, and can be stably expressed in vivo for a long time.

Drawings

The present disclosure may be more fully understood with reference to the following drawings.

FIG. 1 shows a schematic structural diagram of ssAAV-XMRN01 carrier.

FIG. 2 shows a schematic structural diagram of ssAAV-XMRN02 carrier.

FIG. 3 shows H & E staining results after injection of AAV-XMRN01, AAV-XMRN 02. Among them, fig. 3A shows the result at week 6 after injection, and fig. 3B shows the result at week 8 after injection.

FIG. 4 shows a statistical plot of the results of the epiretinal nuclear layer thickness measurements after injection of AAV-XMRN01, AAV-XMRN 02. Among them, fig. 4A shows the result at 6 weeks after injection, and fig. 4B shows the result at 8 weeks after injection.

FIG. 5 shows a statistical plot of nuclear density measurements after injection of AAV-XMRN, AAV-XMRN 02. Among them, fig. 5A shows the result at week 6 after injection, and fig. 5B shows the result at week 8 after injection.

FIG. 6 shows cone immunofluorescent staining results after injection of AAV-XMRN01, AAV-XMRN 02. Among them, fig. 6A shows the result at 6 weeks after injection, and fig. 6B shows the result at 8 weeks after injection.

FIG. 7 shows the results of rod immunofluorescent staining after injection of AAV-XMRN01, AAV-XMRN 02. Among them, fig. 7A shows the result at week 6 after injection, and fig. 7B shows the result at week 8 after injection.

FIG. 8 shows a statistical plot of cone cell numbers after injection of AAV-XMRN, AAV-XMRN 02. Among them, fig. 8A shows the result at week 6 after injection, and fig. 8B shows the result at week 8 after injection.

FIG. 9 shows a statistical plot of rod extracellular segment thickness after injection of AAV-XMRN01, AAV-XMRN 02. Among them, fig. 9A shows the result at week 6 after injection, and fig. 9B shows the result at week 8 after injection.

FIG. 10 shows cone immunofluorescent staining results after injection of AAV-XMRN 03. Wherein, fig. 10A shows an immunofluorescent staining chart of cone cells at week 8 after injection, and fig. 10B shows a statistical chart of cone cell numbers at week 8 after injection.

FIG. 11 shows the results of rod immunofluorescent staining after injection of AAV-XMRN 03. Wherein fig. 11A shows a rod immunofluorescent staining chart at week 8 after injection, and fig. 11B shows a statistical chart of rod extracellular segment thickness at week 8 after injection.

Fig. 12 shows experimental statistics of the light and dark box after injection XMRN a 01. Wherein, fig. 12A shows the bright-dark box statistics at week 5 after injection, and fig. 12B shows the bright-dark box statistics at week 7 after injection.

Figure 13 shows the experimental statistics of the light and dark boxes at week 7 after injection XMRN 03.

Detailed Description

The following description of the present disclosure is intended only to illustrate various embodiments of the present disclosure. Therefore, the particular modifications discussed should not be construed as limiting the scope of the present disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the disclosure, and it is to be understood that such equivalent embodiments are intended to be included herein. All references cited herein, including publications, patents, and patent applications, are incorporated by reference in their entirety.

The numerical limits or ranges stated herein include the endpoints, and specifically include all values and subranges within the numerical limits or ranges.

The present disclosure provides the use of neuropeptide Y (NPY) or a composition containing neuropeptide Y (NPY) in the manufacture of a medicament for treating photoreceptor cell degeneration such as Retinitis Pigmentosa (RP), retinal dystrophy or degeneration, rod cone dystrophy, and the like in a subject.

The present disclosure provides the use of a nucleic acid encoding neuropeptide Y (NPY), a construct or vector system comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY), or a composition comprising the same, in the manufacture of a medicament for treating photoreceptor cell degeneration such as Retinitis Pigmentosa (RP), retinal dystrophy or degeneration, rod cone dystrophy, or the like in a subject.

In another aspect, the present disclosure provides a method of treating photoreceptor cell degeneration such as Retinitis Pigmentosa (RP), retinal dystrophy or degeneration, rod cone dystrophy, or the like in a subject by administering to the subject a therapeutically effective amount of neuropeptide Y (NPY) or a composition containing neuropeptide Y (NPY), a nucleic acid encoding neuropeptide Y (NPY), a construct comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY).

In another aspect, the present disclosure provides the use of neuropeptide Y (NPY) or a composition containing neuropeptide Y (NPY), a nucleic acid encoding neuropeptide Y (NPY), a construct comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY) in a method of treating photoreceptor cell degeneration such as Retinitis Pigmentosa (RP), retinal dystrophy or degeneration, rod cone dystrophy, or the like in a subject.

In some embodiments, the construct contains a nucleic acid encoding neuropeptide Y (NPY). The construct may be constructed by inserting a nucleic acid encoding neuropeptide Y (NPY) into a suitable expression vector, which may be selected by one of skill in the art.

In some embodiments, the cell comprising a nucleic acid encoding neuropeptide Y (NPY) may be a host cell comprising a construct as described above or a nucleic acid as described above integrated into the genome as described above as exogenous or comprising a virus as described in any of the above, such as an adeno-associated virus. As representative examples of suitable host cells, mammalian cells (e.g., CHO or COS), plant cells, human cells (human embryonic kidney cells such as HEK293 FT), bacterial cells (e.g., E.coli, streptomyces, salmonella typhimurium), fungal cells (e.g., yeast), insect cells (e.g., sf 9), etc. can be exemplified. One skilled in the art can select an appropriate host based on the teachings herein. Preferably, the host cell is an animal cell, and more preferably a human cell. The host cell may be a cultured cell or a primary cell, i.e., isolated directly from an organism (e.g., a human). The host cell may be an adherent cell or a cell in suspension, i.e., a cell grown in suspension. Further, the adeno-associated virus also includes a heterologous nucleic acid sequence encoding a product of interest, which may be carried in a coat of various capsid proteins. The heterologous nucleic acid sequence encoding a product of interest described above may typically be a construct, which may typically contain a nucleic acid encoding a product of interest. Such constructs may be generally obtained by inserting a nucleic acid encoding the product of interest into a suitable expression vector, which may be selected by one of skill in the art, such as, but not limited to, pAAV-CAG, pAAV-TRE, pAAV-EF1a, pAAV-GFAP promoter, pAAV-Lgr5 promoter, pAAV-sox2 promoter expression vectors, and the like. In the present disclosure, when the adeno-associated virus encodes a heterologous nucleic acid sequence for a product of interest, the adeno-associated virus contains a capsid, and the viral vector carries a transgene encoding a gene product, which transgene is under the control of regulatory sequences directing its expression in a host cell.

In some embodiments, the disclosure also provides an engineered host cell obtained using adeno-associated virus transformation as described above. The engineered host cell comprises the adeno-associated virus described above. The host cell may be eukaryotic and/or prokaryotic.

In some embodiments, the cell comprising the nucleic acid encoding neuropeptide Y (NPY) may also be a therapeutic cell, which is introduced into the subject, and thereby expresses neuropeptide Y (NPY) in the subject, to achieve a therapeutic effect.

In some embodiments, the virus of the nucleic acid encoding neuropeptide Y (NPY) may be an adeno-associated virus. The adeno-associated virus comprises a capsid, the serotype of which is selected from AAV2, AAV5 or AAV9. In some embodiments, the serotype of the AAV is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8R, AAV, AAV10, or AAVrh10. In some embodiments, the AAV vector comprises an AAV Inverted Terminal Repeat (ITR) sequence flanked by one or more AAV Inverted Terminal Repeat (ITR) sequences. In some embodiments, the heterologous nucleic acid is flanked by two AAV ITRs. In some embodiments, the AAV ITRs are ITRs of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8R, AAV9, AAV10, AAVrh10 serotypes. In some embodiments, the AAV ITRs are AAV2 ITRs. In some embodiments, the ITR and the capsid of the AAV particle are derived from the same AAV serotype. In some embodiments, the ITR and the capsid are derived from AAV2. In other embodiments, the ITRs and capsids of the AAV viral particles are derived from different AAV serotypes. In the present invention, the term "AAV" is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The term "AAV virus" or "AAV viral particle" or "AAV vector particle" refers to a viral particle of an AAV vector comprising at least one AAV capsid protein and one encapsulated polynucleotide.

In some embodiments, the vector comprising the encoded NPY may use any existing expression and/or delivery vector that may be used for NPY, such as at least one selected from AAV vectors, adenovirus vectors, lentiviral vectors, RNA viral vectors, or vaccinia viral vectors.

In some embodiments, the vector comprising the encoded NPY further comprises at least one of the following functional elements: inverted Terminal Repeat Sequences (ITRs), promoters, regulatory elements and polyA sequences. The regulatory element may be, for example, a cytomegalovirus enhancer, an Internal Ribosome Entry Site (IRES), a woodchuck hepatitis posttranslational regulatory element (WPRE), and the like. Such as the promoter of the ampicillin resistance gene, the kanamycin resistance gene, the Cytomegalovirus (CMV) promoter, the Cytomegalovirus (CMV) enhancer-chicken beta-actin (CAG) promoter, the cytomegalovirus enhanced/beta-actin (CMV ENHANCER/beta-actin, CB) promoter, the neuron-specific human synopsin 1 (SYN) promoter, and the astrocyte-specific GfaABC D/GFAP promoter. The polyA sequence may be, for example, a bovine growth hormone polyadenylation (bGH-polyA) signal sequence.

In some embodiments, the AAV vector comprises one or more promoters, and/or enhancers, and/or polyadenylation signals.

In some preferred embodiments, the vector is an adeno-associated viral vector comprising:

1) A promoter;

2) Nucleic acid encoding neuropeptide Y (NPY);

3) One or both ITR sequences, preferably ITR sequences of AAV2 virus.

In some embodiments, the vector system of the present disclosure may be an adeno-associated virus vector system comprising a packaging plasmid further comprising therein a rep gene fragment of an adeno-associated virus. Wherein the rep gene fragment comprises an intron comprising a transcription termination sequence. The packaging plasmid, the expression plasmid and the helper virus plasmid are transferred into a host cell, and the nucleic acid sequences of the packaging plasmid, the expression plasmid and the helper virus plasmid are all integrated in the host cell to produce the adeno-associated virus. In some embodiments, the nucleic acid sequences are all integrated together at a single locus within the cell genome of the host cell. In some embodiments, the nucleic acid sequences encoding the various genes are present as separate expression cassettes; the nucleic acid sequences encoding the rep and cap genes are present in the same or different expression cassettes.

In some embodiments, as shown in FIGS. 1 and 2, the adeno-associated viral vector comprises an ITR sequence, a nucleic acid encoding NPY, a promoter, a kanamycin resistance gene (KanR) gene, an ori gene, and an f1 ori gene.

The term "neuropeptide Y"

Neuropeptides Y (Neuropeptide Y, NPY) belong to the NPY family of neuroendocrine peptides, which also includes peptide YY (PEPTIDE YY, PYY) and pancreatic polypeptide (PANCREATIC POLYPEPTIDE). NPY is widely present throughout the body. Neuropeptides Y (NPY) and NPY receptors are widely expressed in the central nervous system, including the retina. Retinal cells, in particular neurons, astrocytes and muller cells, microglia and endothelial cells express this peptide and its receptor (Y1, Y2, Y4 and/or Y5).

In some embodiments, the neuropeptide Y in the present disclosure is a mammalian-derived NPY or variant thereof, preferably a human-derived NPY (hNPY), a human-derived material NPY (hmNPY), a rat-derived NPY (rNPY), a mouse-derived NPY (musNPY), or variant thereof.

Specifically, a variant of NPY refers to a polypeptide fragment obtained by substituting, deleting or adding one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1,2, or 3) amino acids to the amino acid sequence of NPY, or adding one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1,2, or 3) amino acids to the N-terminus and/or the C-terminus, but still having NPY function. The variant may have at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an NPY such as any of SEQ ID NO. 1 or 3.

In some embodiments, the hNPY comprises an amino acid sequence selected from SEQ ID No. 1, having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 1.

In some embodiments, the nucleic acid encoding the hNPY comprises a nucleic acid sequence selected from SEQ ID No. 2 having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 2.

In some embodiments, the hmNPY comprises an amino acid sequence selected from SEQ ID NO. 3, having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 3.

In some embodiments, the nucleic acid encoding the hmNPY comprises a nucleic acid sequence selected from SEQ ID NO. 4, having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 4.

In some embodiments, the rNPY comprises an amino acid sequence selected from SEQ ID NO. 5, having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 5.

In some embodiments, the nucleic acid encoding rNPY comprises a nucleic acid sequence selected from SEQ ID NO. 6, having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 6.

In some embodiments, the musNPY comprises an amino acid sequence selected from SEQ ID NO. 7, having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 7.

In some embodiments, the nucleic acid encoding musNPY comprises a nucleic acid sequence selected from SEQ ID NO. 8, having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 8.

In some embodiments, the composition comprising neuropeptide Y (NPY) further comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may include various carriers, excipients, diluents, and the like, which are not per se essential active ingredients, and which are not excessively toxic after administration. Suitable vectors should be well known to those skilled in the art. Such as sterile or normal saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphoric acid, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, tween, etc.), chelating agents (EDTA, etc.), binders, etc. Furthermore, other low molecular weight polypeptides may be included; proteins such as serum albumin, gelatin, and immunoglobulins; amino acids such as glycine, glutamine, asparagine, arginine and lysine; saccharides or carbohydrates such as polysaccharides and monosaccharides; sugar alcohols such as mannitol and sorbitol. When preparing an aqueous solution for injection, such as physiological saline, isotonic solution containing glucose or other auxiliary drugs, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and the like, an appropriate solubilizing agent such as alcohol (ethanol or the like), polyol (propylene glycol, PEG or the like), nonionic surfactant (Tween 80, HCO-50) or the like may be used in combination.

In some embodiments, a second agent that can treat photoreceptor cell degeneration is further included in the composition. The second drug is packaged with neuropeptide Y (NPY) in the same or different medium or kit. In some embodiments, the second agent may be administered before or after or simultaneously with the administration of neuropeptide Y (NPY), a nucleic acid encoding neuropeptide Y (NPY), a vector comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY).

In some embodiments, adeno-associated viruses or pharmaceutical compositions and the like provided by the present disclosure may be adapted for administration by subcutaneous, intramuscular, articular, nervous, or circulatory systems such as blood or body fluids, in a suitable manner of administration. Such as the composition is suitable for intravenous or intraocular administration. The skilled artisan can select the appropriate dosage depending on the mode of administration. In some embodiments, the drug is administered by intravitreal injection.

The amount of the active ingredient of the adeno-associated virus or pharmaceutical composition to be administered is generally dependent on the weight of the patient, the type of application, the condition and severity of the disease, such as the amount of the virus or pharmaceutical composition to be administered as an active ingredient may be generally 1 to 1000 mg/kg/day, 1 to 3 mg/kg/day, 3 to 5 mg/kg/day, 5 to 10 mg/kg/day, 10 to 20 mg/kg/day, 20 to 30 mg/kg/day, 30 to 40 mg/kg/day, 40 to 60 mg/kg/day, 60 to 80 mg/kg/day, 80 to 100 mg/kg/day, 100 to 200 mg/kg/day, 200 to 500 mg/kg/day, or more than 500 mg/kg/day.

Definition of the definition

Before further describing the present invention, certain terms used in the specification, examples, and appended claims are collected in the following sections. The definitions set forth herein should be read and understood by those skilled in the art in light of the remainder of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. With respect to the use of virtually any plural and/or singular terms herein, one of skill in the art can translate from the plural to the singular and/or from the singular to the plural depending on the context and/or application. For clarity, various singular/plural arrangements may be explicitly set forth herein.

As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

The terms "comprising," "including," and the like are to be construed as inclusive and not exclusive or exhaustive; i.e., the meaning of "including but not limited to".

The nucleic acid or polynucleotide of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The nucleic acids or polynucleotides of the invention may also be variants of the above-described nucleic acids or polynucleotides which encode polypeptides or fragments, analogs and derivatives of polypeptides with the amino acid sequences required in the invention. The variant of the nucleic acid or polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. Such nucleic acid or polynucleotide variants include substitution variants, deletion variants, and insertion variants. As known in the art, an allelic variant is a substitution pattern of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the polypeptide it encodes.

The term "amino acid" includes any naturally occurring amino acid, modified forms thereof, and synthetic amino acids. Such modified forms include amino acid residues modified or amino acids modified by post-translational modifications such as acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation or sulfation.

The term "nucleic acid construct" refers herein to a DNA or RNA molecule, which contains a nucleotide sequence encoding NPY. The coding sequence includes initiation and termination signals operably linked to regulatory elements, and may also include promoters and polyadenylation signals capable of directing expression in cells of the individual receiving the nucleic acid molecule.

Adeno-associated viruses (AAV) are small, naturally occurring, non-pathogenic viruses belonging to the genus of dependoviridae in the parvoviridae family. Although not causing disease, AAV is known to be capable of infecting humans and other primates, and is prevalent in the human population. A variety of serotypes of AAV have been identified (such as AAV2, AAV5, AAV6, etc.), which exhibit different tissue infectivity. AAV is a single stranded DNA virus, consisting of approximately 4,800 nucleotides. AAV genomes include rep genes, capsid (cap) genes, AAP genes, and the like. The Rep gene encodes four nonstructural Rep proteins (Rep 40, rep52, rep68, and Rep 78) by using multiple reading frames, staggered promoters (P5, P19, and P40), and alternative splicing, which are required for viral transcriptional control and replication and packaging of the virus into viral capsids. The capsid gene encodes three capsid proteins (VPs): VP1, VP2, and VP3. These three capsid proteins are translated from a single mRNA transcript controlled by a single promoter (P40 in the case of AAV 2). The AAP gene encodes AAV Assembly Activating Protein (AAP). Sixty VP monomers (including about 5 copies of VP1, 5 copies of VP2, and 50 copies of VP 3) self-assemble around the AAV genome to form the icosahedral protein shell (capsid) of the mature viral particle. The AAV gene coding sequence is flanked by two AAV-specific palindromic inverted repeats (ITRs) of 145 nucleotides. AAV is a naturally defective virus, lacking the ability to perform at least two vital functions: the ability to initiate synthesis of virus-specific products and the ability to assemble such products to form mature infectious viral particle capsids. Thus, AAV requires co-infection with a helper virus, such as adenovirus (Ad), herpes Simplex Virus (HSV), cytomegalovirus (CMV), vaccinia virus, or human papillomavirus, to provide a virus-associated (VA) RNA that is not encoded by the genes of the AAV genome. Such VA RNAs do not undergo translation but play a role in regulating translation of other viral genes. In addition, the capsid or genomic element of an AVV virus may contain other modifications, including insertions, deletions, and/or substitutions.

RAAV is typically produced using circular plasmids ("rAAV plasmid vectors"). The AAVrep and cap genes are typically deleted from such constructs and replaced with promoters, beta globulin introns, cloning sites for insertion of selected therapeutic genes (transgenes), and polyadenylation ("polyA") sites. However, the terminal inverted repeat (ITR) of AAV is still retained, such that the transgene expression cassette of the rAAV plasmid vector is flanked by ITR sequences. Thus, in the 5 'to 3' direction, rAAV includes a 5'itr, a transgenic expression cassette for rAAV, and a 3' itr.

The term "inverted terminal repeat" or "ITR" includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and serves as an inverted terminal repeat (i.e., mediates a desired function, such as replication, viral packaging, integration, and/or proviral rescue, etc.). The ITRs can be AAV ITRs or non-AAV ITRs. For example, non-AAV ITR sequences, such as those of other viruses (e.g., canine small virus (CPV), mouse small virus (MVM), human small virus B-19) or any other suitable viral sequences (e.g., SV40 hairpin that serves as the origin of SV40 replication) may be used as ITRs, which may be further modified by truncation, substitution, deletion, insertion, and/or addition. Furthermore, the ITRs can be partially or fully synthetic.

An "AAV inverted terminal repeat" or "AAV ITR" may be from any AAV, including but not limited to serotypes 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or any other AAV currently known or subsequently discovered. AAV inverted terminal repeats need not have native terminal repeats (e.g., the native AAV ITR sequences can be altered by insertions, deletions, truncations, and/or missense mutations), so long as the terminal repeats mediate a desired function, e.g., replication, viral packaging, integration, and/or proviral rescue, etc.

The term "disease" as used herein is generally synonymous with the terms "disorder", "syndrome" and "condition" and is used interchangeably as they both reflect an abnormal condition of one of the human or animal body or parts thereof that impairs normal function, often manifests as identifying signs and symptoms, and results in a reduced life span or quality of life of the human or animal.

The term "treatment" is to be understood in its broadest sense, including both therapeutic and prophylactic measures, wherein the aim is to prevent or slow down (alleviate) an undesired physiological condition, disorder or disease, or to obtain a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of the disorder, condition or disease, stabilized (i.e., not worsening) state of the disorder, condition or disease, delay of onset or slowing of progression of the disorder, condition or disease, amelioration of the state of the disorder, condition or disease, and diminishment of any of the detectable or undetectable remission (whether partial or total) or facilitation or amelioration of the disorder, condition or disease. Treatment involves eliciting a clinically significant response without excessive levels of side effects. Treatment also includes extending survival compared to expected survival without treatment. Treatment may also be pre-established in nature, i.e., it may include prevention of disease. Prevention of a disease may involve complete protection from the disease, for example in the case of preventing infection by a pathogen, or may involve preventing disease progression. For example, prevention of a disease may not mean complete loss of any effect associated with the disease at any level, but may mean prevention of symptoms of the disease to clinically significant or detectable levels. Prevention of a disease may also mean preventing the disease from progressing to a later stage of the disease. As used herein, "treatment" encompasses treatment of a disease (primarily neurodegenerative disease) in a mammal, particularly a human, including: (a) Preventing the occurrence of a disease (e.g., preventing a neurodegenerative disease) or disorder in an individual susceptible to the disease but not yet diagnosed with the disease; (b) inhibiting disease, e.g., arresting disease progression; or (c) alleviating a disease, e.g., alleviating symptoms associated with a disease. As used herein, "treating" or "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in the individual, including, but not limited to, administration of a drug comprising lipoic acid and crataegus described herein to an individual in need thereof.

The term "therapeutically effective amount" generally means an amount which, after a suitable period of administration, achieves the effect of treating the diseases as set forth above. The actual amount comprising an "effective amount" will vary depending upon a variety of circumstances including, but not limited to, the severity of the disease, the size and health of the patient, the imaging modality, the diagnostic modality, the monitoring modality, and the route of administration. The appropriate amount can be readily determined by a skilled medical practitioner using methods known in the medical arts.

The terms "patient" and "subject" are interchangeable and can mean any living organism that can be treated with the pharmaceutical compositions of the present disclosure. In the present disclosure, the subject or individual undergoing therapeutic or prophylactic treatment is preferably a mammal, such as, but not limited to, a human, a non-human primate, livestock (e.g., sheep, cow, horse, donkey, pig), companion animals (e.g., dogs, cats), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), or captured wild animals (e.g., foxes, deer). The subject is preferably a primate. In certain embodiments, the subject is a mammal, preferably the subject is a human. In certain embodiments, the subject is an adult, child, or infant.

The term "administration" as used herein refers to the administration of a pharmaceutical composition directly to the subject by a physician or health care provider.

The particular mode of administration will depend on the indication or purpose. The choice of the particular route of administration and dosage regimen will be adjusted or titrated by the medical practitioner according to methods known in the medical arts to obtain the optimal clinical response. The amount of the compound to be administered is an effective amount. The dose to be administered will depend on the characteristics of the subject being treated, such as the particular animal being treated, the age, weight, health, the type of concurrent treatment (if any) and the frequency of treatment, and can be readily determined by one of skill in the art (e.g., by a clinician).

Before the embodiments of the present disclosure are further described, it is to be understood that the scope of the disclosure is not limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples of the present disclosure is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present disclosure; in the present disclosure and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Where numerical ranges are given in the examples, it is understood that unless otherwise indicated by the present disclosure, both endpoints of each numerical range and any number between the endpoints are significant both are alternatives. Unless defined otherwise, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art that are similar or equivalent to those described in the embodiments of the present disclosure may be used to implement the present disclosure, based on the knowledge of one skilled in the art and the recitation of the present disclosure.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present disclosure employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts. These techniques are well described in the prior art.

Statistical analysis

Data processing and statistical analysis were performed using GRAPHPAD PRISM 8.0.0 software. The statistical level is set at 5% or p is less than or equal to 0.05, the average number and standard error (mean+/-SEM) of each analysis index are calculated, and p is less than or equal to 0.05, so that the difference has statistical significance.

In order that those skilled in the art will better understand the present disclosure, a technical solution in the embodiments of the present disclosure will be clearly and completely described below, and it is apparent that the described embodiments are only some embodiments of the present disclosure, not all embodiments.

Examples

EXAMPLE 1 construction of plasmid vector

Construction of AAV vector plasmids expressing human NPY (hNPY), human material NPY (hmNPY) and rat NPY (rNPY) genes

According to the amino acid sequence of hNPY (P01303) published by UniProt (SEQ NO:1, see Table 1), the nucleotide sequence was designed according to human codon preference (SEQ NO: 2), a BamHI cleavage site was introduced at the 5 'end and an EcoR V cleavage site was introduced at the 3' end, and the resulting nucleic acid construct was designated XMRN01. Wherein, the target gene is synthesized by Beijing qing department biotechnology Co., ltd, bamH I and EcoR V are adopted to digest XMRN and ssAAV plasmids, and then the ssAAV-XMRN01 vector is constructed by the conventional molecular biology techniques of T4 ligase (T4 DNALIGASE), transformation, monoclonal screening and the like, and the structural diagram is shown in figure 1. After double verification of PCR and sequencing, the positive clone was subjected to plasmid DNA preparation using endotoxin-free plasmid extraction kit (MACHEREY-NAGEL GmbH & Co.KG, cat. No. 740420.50) for use.

The nucleotide sequence was designed according to the human codon preference (SEQ NO:4, see Table 1) based on the published amino acid sequence of hNPY on UniProt (SEQ NO:1, see Table 1) and the amino acid sequence of hmNPY reported by Ana Santos-Carvalho et al (SEQ NO:3, see Table 1), with a BamH I cleavage site introduced at the 5 'end and an EcoR V cleavage site introduced at the 3' end, and the resulting nucleic acid construct was designated XMRN. Wherein, the target gene is synthesized by Beijing qing department biotechnology Co., ltd, bamH I and EcoR V are adopted to digest XMRN and ssAAV plasmids (the same as above), and then the ssAAV-XMRN02 vector is constructed by conventional molecular biology techniques such as T4 ligase (T4 DNA LIGASE), transformation, monoclonal screening and the like, and the structural schematic diagram is shown in figure 2. Positive clone is subjected to double verification of PCR and sequencing, and high-quality plasmid DNA is obtained for standby by using an endotoxin-free plasmid extraction kit (the same as above).

A vector ssAAV-XMRN was constructed by the method described above based on the amino acid (SEQ ID NO:5, see Table 1) and nucleic acid (SEQ ID NO:6, see Table 1) sequences of rNPY (P07808) published by UniProt. Positive clone is subjected to double verification of PCR and sequencing, and high-quality plasmid DNA is obtained for standby by using an endotoxin-free plasmid extraction kit (the same as above).

The nucleic acid sequence (SEQ ID NO:10, see Table 1) was designed according to human codon preference based on the amino acid sequence (SEQ ID NO:9, see Table 1) of eGFP (A0A 076FL 24) published on the UniProt database, and was introduced with a BamH I cleavage site at the 5 'end and an EcoR V cleavage site at the 3' end, designated NC. The gene is synthesized by Beijing Qingqike biotechnology Co., ltd, and after digestion of NC and ssAAV plasmids with BamH I and EcoR V, a ssAAV-NC vector is constructed by conventional molecular biology techniques such as T4 ligase (T4 DNA LIGASE), transformation and monoclonal screening. Positive clone is subjected to double verification of PCR and sequencing, and high-quality plasmid DNA is obtained for standby by using an endotoxin-free plasmid extraction kit (the same as above).

TABLE 1 sequence information

Example 2 preparation and identification of recombinant AAV viruses

The recombinant AAV virus was prepared using a three-plasmid packaging system, as follows: helper plasmids, AAV Rep and Cap protein expression plasmids and ITR expression plasmids (ssAAV-XMRN 01, ssAAV-XMRN02, ssAAV-XMRN03, ssAAV-NC) constructed in example 1 were used to obtain plasmid DNA for use with endotoxin-free plasmid extraction kits (same as in example 1). HEK293T cells were subcultured in 10cm dishes and, when the confluency reached about 90%, replaced with 4mL of low serum medium (DMEM medium, 4% FBS, 1% penicillin-streptomycin, wherein DMEM medium was purchased from Gibco company, FBS was purchased from Natocor company, penicillin-streptomycin was purchased from Gibco company) for 2 hours.

Preparing transfection reagent: to each dish was added transfection reagent (1 mL Opti-MEM, 40. Mu.g of transfection-promoting agent using polyethylenimine (Polyethyleneimine, PEI, available from Polysciences) and 10. Mu.g of pHelper, 5. Mu.g of AAV, 5. Mu. g XMRN01/XMRN 02/XMRN/NC) respectively. After about 6 hours of transfection, the liquid in the dishes was replaced with 10mL of low serum medium (DMEM medium, 4% FBS, 1% penicillin-streptomycin) and the culture was continued back to the incubator. Viruses in the supernatant were collected in two times: after culturing for 3 days, collecting cell culture supernatant, continuously adding fresh low serum culture medium, placing back into an incubator for continuous culture, centrifuging the cell supernatant at 3000rpm for 10 minutes, and removing cell fragments; after 7 days of culture, cell culture supernatant was collected and centrifuged at 3000rpm for 10 minutes to remove cell debris.

Purification of recombinant AAV virus: firstly, concentrating the cell culture supernatant to about 10mL by adopting a centrifugal filter device (Millipore, cat No. 910024), then obtaining pure recombinant AAV by adopting an iodixanol gradient centrifugation method, eluting by PBS, split charging, and storing in a refrigerator at the temperature of minus 80 ℃ for standby.

Titer determination of recombinant AAV virus: the obtained purified recombinant AAV virus is detected by qPCR method, and then the morphology, titer, purity and the like of the recombinant AAV virus are identified again by a transmission electron microscope. Thus obtaining recombinant AAV virus with qualified titer and purity.

Example 3 efficacy of AAV-XMRN01, AAV-XMRN02, AAV-XMRN03 in an animal model for retinal photoreceptor degeneration

The rod cells of P23H (+/-) mice showed regional degeneration and a decrease in rod outer cell segment (rod outer segment, ROS) length, photoreceptor degenerative changes, decreased retinal thickness, as a model of autosomal retinitis pigmentosa (adRP) associated with human P23H rhodopsin mutations (see Sanae,Sakami,Tadao,et al.Probing Mechanisms of Photoreceptor Degeneration in a New Mouse Model of the Common Form of Autosomal Dominant Retinitis Pigmentosa due to P23H Opsin Mutations[J].Journal of Biological Chemistry,2011.DOI:10.1074/jbc.m110.209759.). these mouse rod photoreceptor degeneration required approximately 4 to 5 months, resulting in significant loss of outer nuclear layer (outer nuclear layer, ONL) and visual function).

The specific method of propagation of P23H (+/-) mice is as follows: adult C57BL/6J females and P23H (+/-) males were caged and the offspring were genotyped and P23H (+/-) mice were screened.

SPF-class 10-day-old (P10) and 28-day-old (P28) P23H (+/-) mice (raised on SPF-class laboratory animal platform of the national center for Care of Care, endoconcha-use license number SYXK (Anhui) 2020-006) were raised under an alternating light and dark environment for 12 hours according to the mouse rhythm, and the weight of 28-day-old mice was about 14g, and the animals with no abnormality in hair, normal eyeballs and acceptable weight were injected by performing a general examination before injection.

Groups were given by intraocular injection on day 1 (D1), behavioral experiments were performed on day 7 (D49) and day 28P 23H (+/-) mice after injection (D35) respectively (see Berry M,Holt A,Salari A,et al.Restoration of high-sensitivity and adapting vision with a cone opsin[J].Investigative ophthalmology&visual science,2019.), for analysis of the effect on the vision of mice after administration. Then, on day 10P 23H (+/-) mice, day 8 (D56) and day 28P 23H (+/-) mice after injection (D42), 3 mice were euthanized each group, eyeballs were frozen and frozen, and retinal structures were analyzed by immunofluorescent staining and H & E staining observation.

TABLE 2 intraocular injection of mice on day 1

The specific operation steps are as follows:

Intravitreal injection administration: placing a 10-day-old mouse on ice for about 3 minutes to achieve an anesthetic effect, and using a needle to scratch the eyelid of the mouse to expose the eyeball; the eyes of the mice of 28 days old are respectively dropped with 5% of mydriatic fluid for mydriatic and intraperitoneal injection of 240mg/kg of aphtha for anesthesia. The anesthetized animals were then placed on their side on a console, and 1-2mm posterior to the temporal or epistaxis limbus of the eye was selected as the injection needle site (syringe: hamilton needle, 7632-01), taking care to avoid damaging the posterior capsule of the lens and other retinal sites. The needle head enters the vitreous cavity, the injection is injected by pushing, the needle is slowly pulled out after stopping for 10 seconds, erythromycin eye ointment is smeared, and the erythromycin eye ointment is put back into the cage.

Subretinal space injection administration: the 10-day-old mice were placed on ice for about 3 minutes to achieve anesthesia, the mice were opened with a needle to expose the eye, then the animals were placed on their sides on a console, and 1-2mm behind the temporal or epistaxis limbus of the eyes were selected as the injection needle site (injector: hamilton needle, 7632-01), taking care to avoid damaging the posterior capsule of the crystals and other retinal sites. The needle head enters the subretinal cavity, the injection is injected in a pushing way, and after the injection is finished, the needle is slowly pulled out, erythromycin eye ointment is coated, and the erythromycin eye ointment is put back into the cage.

Example 4: hematoxylin eosin (H & E) staining to assess the effects of AAV-XMRN01, AAV-XMRN02 on the thickness of the outer nuclear layer and nuclear density of the retina of P23H (+/-) model mice

The mice in example 3 were enucleated, unilateral eyes were fixed overnight at 4% paraformaldehyde (Biosharp) at 4 ℃, the cornea was peeled off from the lens after 4 washes with PBS (Biosharp), eye cups such as retina, choroid, sclera, etc. were left to be dehydrated by 30% sucrose (biochemical) until 10 μm thick frozen sections were made after sinking, the sections were H & E stained, photographed after staining was completed and the retinal sections were measured for Outer Nuclear Layer (ONL) thickness and nuclear density with Image J and the differences were analyzed.

Results of H & E staining, epiretinal nuclear layer thickness, and nuclear density at week 6 after injection in 28 day-old P23H (+/-) mice are shown in FIGS. 3A, 4A, and 5A, respectively; h & E staining, epiretinal nuclear layer thickness, and nuclear density results at week 8 after injection of 10 day-old P23H (+/-) mice are shown in FIGS. 3B, 4B, and 5B, respectively. Compared with the respective control group, the thickness of the outer nuclear layer and the nuclear density of the retina of the model mice are obviously reduced for 2 mice injected at different ages; compared with a model group, the injection of AAV-XMRN01 can obviously increase the thickness and the nuclear density of the outer nuclear layer, improve the structural degradation of retina, and the injection of AAV-XMRN02 has the trend of increasing the thickness and the nuclear density of the outer nuclear layer; AAV-XMRN01 has better effect on improving the membrane thickness and nuclear density than AAV-XMRN02.

Example 5: immunofluorescent staining to assess the effects of AAV-XMRN01, AAV-XMRN02, AAV-XMRN03 on P23H (+/-) model mouse retinal photoreceptor cells

Air-drying the frozen sections of example 4 in a slide box at room temperature, and circling the tissues on the slide with an immunohistochemical pen (Vectorlabs); placing a polylysine slide glass (Shitai) into a wet box, washing tissues on the slide glass by using PBS, incubating for 5 minutes at room temperature, pouring out the PBS on the slide glass, and washing for 5 times each time for 10 minutes; adding 500 μl/piece of blocking solution (5% goat serum (Biyun day) +0.5% Triton 100 (Ind.)) and incubating in a wet box for 45 min; pouring off the sealing liquid on the glass slide, and dripping the primary antibody mixed liquid: cone cell sensitive material Cone Arresting-1 (EMD Millipore Corp) and rod cell sensitive material Rodopsin-1 (santa cruz), the dilution ratio was 1:500, 200. Mu.L/sheet, placed in a wet box and incubated overnight at 4 ℃; the next day, wash with PBS 5 times at room temperature for 5 minutes each; adding a secondary antibody mixed solution: anti-rabbit IgG Alexa Fluor 555 (CELL SIGNALING) and Donkey anti-mouse IgG (H+L) Highly Cross-Adsorbed Alexa Fluor 488 (Invitrogen) were counterstained at 1:500, incubated in a wet box in the dark at room temperature for 2.5 hours; light shielding is needed after the step; removing the secondary antibody, and washing with PBS at room temperature for 5 times, each time for 5 minutes; DAPI staining solution (Sigma) was added, 50. Mu.L/sheet, incubated at room temperature for 10 minutes, and then rinsed 3 times with PBS for 5 minutes each; and sealing the piece, and observing and photographing under a fluorescence microscope.

6 Weeks after the vitreous cavity injections XMRN and XMRN02 of the 28 day old P23H (+/-) mice, the results of cone immunofluorescent staining, rod immunofluorescent staining, statistics of the number of cone cells, and statistics of the thickness of the rod extracellular segment are shown in FIGS. 6A, 7A, 8A, and 9A, respectively; the results of cone immunofluorescent staining, rod immunofluorescent staining, statistics of cone number, and rod extracellular segment thickness at week 8 after vitreous cavity injections XMRN and XMRN of 10 day old P23H (+/-) mice are shown in FIGS. 6B, 7B, 8B, and 9B, respectively. Compared with a control group, the number of retina cone cells in the model group is reduced, the thickness of the extracellular segment of the rod is narrowed, and the photoreceptor cells are obviously degenerated; compared with the model group, the injection of AAV-XMRN01 and AAV-XMRN02 can obviously improve the degeneration of cone cells and rod cells.

The results of cone immunofluorescent staining, rod immunofluorescent staining, statistics of cone number, and rod extracellular segment thickness at week 8 after injection XMRN in the subretinal space of the 10 day old P23H (+/-) mice are shown in FIGS. 10 and 11, respectively. Compared with the model group, the AAV-XMRN03 injection can obviously improve the degeneration of the cone cells and the rod cells.

Example 6: behavioural assessment of the effects of AAV-XMRN01, AAV-XMRN02, AAV-XMRN03 on the vision of P23H (+/-) model mice

The light and dark box experiments were performed at week 7 (D49) after injection of 10 day old P23H (+/-) mice and at week 5 (D35) after injection of 28 day old P23H (+/-) mice, respectively, as described in example 3. The bright and dark box is a box body, the bright area is a transparent area, the dark area is a black area, the volume of the dark area is one half of that of the bright area, the illumination intensity is far lower than that of the bright area, and a small door is connected with the bright and dark area, so that animals can freely pass through the bright and dark area. This test relies on the natural preference of mice for the lighting environment and the vision situation, and normal vision mice tend to stay in the dark for longer periods. The mice are adapted to 2h in advance between behaviours in a dark way, then are put into a bright and dark box for adaptation for 10 minutes, then the actions of the mice in the bright area and the dark area respectively in 10 minutes are shot and recorded, the stay time of each area is counted, the time of the mice in the bright box is calculated, and the influence on the vision of the mice after administration is analyzed according to the time ratio (Berry M,Holt A,Salari A,et al.Restoration of high-sensitivity and adapting vision with a cone opsin[J].Investigative ophthalmology&visual science,2019.),.

FIG. 12A shows the results of the light and dark box statistics at week 5 after the vitreous cavity injection XMRN of the 28 day old P23H (+/-) mice; the 7 th week dark and light box statistics after the vitreous cavity injection XMRN of the 10 day old P23H (+/-) mice are shown in FIG. 12B. Compared with a control group, the model group stays in the bright box for a long time and is relatively insensitive to illumination, so that the vision degradation is demonstrated; injection of AAV-XMRN01 improved vision deterioration compared to the model group.

The 7-week-old light and dark box statistics after the subretinal space injection XMRN for the 10-day-old P23H (+/-) mice are shown in FIG. 13. Injection of AAV-XMRN03 improved vision deterioration compared to the model group.

Incorporated by reference

The entire contents of each patent and scientific document referred to herein is incorporated by reference for all purposes.

Equivalency of

The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. The above embodiments should therefore be regarded as illustrative in all respects, rather than limiting on the invention described herein. The scope of the disclosure is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims (10)

1. Use of neuropeptide Y (NPY), a pharmaceutical composition comprising neuropeptide Y (NPY), a nucleic acid encoding neuropeptide Y (NPY), a vector comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY), or a pharmaceutical composition comprising the same, in the manufacture of a medicament for treating photoreceptor cell degeneration in a subject, preferably comprising at least one of retinal pigment degeneration (RP), retinal dystrophy or degeneration, rod cone dystrophy.

2. Use according to claim 1, wherein the neuropeptide Y is NPY of mammalian origin, preferably human neuropeptide Y (hNPY) or a mature human neuropeptide Y (hmNPY), rat neuropeptide Y (rNPY), mouse neuropeptide Y (musNPY).

3. The use of claim 2, wherein the hNPY comprises an amino acid sequence selected from SEQ ID No.1 having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 1;

The nucleic acid encoding the hNPY comprises a nucleic acid sequence selected from SEQ ID No. 2 having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 2;

Said hmNPY comprises an amino acid sequence selected from SEQ ID No. 3, having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 3;

The nucleic acid encoding said hmNPY comprises a nucleic acid sequence selected from the group consisting of SEQ ID No. 4, having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 4;

The rNPY comprises the amino acid sequence shown as SEQ ID NO. 5, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 5;

The nucleic acid encoding said rNPY comprises a nucleic acid sequence selected from the group consisting of SEQ ID No. 6, having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 6;

the musNPY comprises the amino acid sequence shown as SEQ ID NO. 7, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 7;

The nucleic acid encoding said rNPY comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO. 8, having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 8.

4. Use according to claim 2 or 3, wherein the vector comprising the encoded NPY further comprises at least one of the following functional elements: inverted Terminal Repeat Sequences (ITRs), promoters, regulatory elements and polyA sequences.

5. The use according to any one of claims 2 to 4, wherein the vector comprising the encoded NPY is selected from at least one of an adeno-associated viral AAV vector, an adenovirus vector, a lentiviral vector, an RNA viral vector or a vaccinia viral vector.

6. The use of any one of claims 2 to 5, wherein the virus comprising a nucleic acid encoding NPY is an AAV virus, preferably wherein the serotype of AAV capsid proteins in the AAV virus is selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8R, AAV, AAV10, AAVrh10, or AAVDJ.

7. The use of any one of claims 1 to 6, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

8. The use of any one of claims 1 to 7, wherein the pharmaceutical composition further comprises a second medicament for treating photoreceptor cell degeneration.

9. The use of any one of claims 1 to 8, wherein the subject is a mammalian subject, preferably a human subject.

10. The use of any one of claims 1 to 9, wherein the medicament is suitable for intraocular, intravenous, intra-arterial, subcutaneous, intramuscular, intratracheal or inhalation administration.