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WO2025214408A1 - Modulation optogénétique pour restauration de la vision - Google Patents

Modulation optogénétique pour restauration de la vision

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Publication number
WO2025214408A1
WO2025214408A1 PCT/CN2025/088056 CN2025088056W WO2025214408A1 WO 2025214408 A1 WO2025214408 A1 WO 2025214408A1 CN 2025088056 W CN2025088056 W CN 2025088056W WO 2025214408 A1 WO2025214408 A1 WO 2025214408A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
nucleotide sequence
polynucleotide construct
raav
opn5
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/088056
Other languages
English (en)
Inventor
Yu Wu
Yanqun Shu
Jinzhao Hou
Yanjun SHEN
Shihua CAO
Lingling ZHAO
Jinxin DENG
Bing Wu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Skyline Therapeutics Shanghai Co Ltd
Skyline Therapeutics Ltd
Original Assignee
Skyline Therapeutics Shanghai Co Ltd
Skyline Therapeutics Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Skyline Therapeutics Shanghai Co Ltd, Skyline Therapeutics Ltd filed Critical Skyline Therapeutics Shanghai Co Ltd
Publication of WO2025214408A1 publication Critical patent/WO2025214408A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA

Definitions

  • the present disclosure relates to molecular biology.
  • the present disclosure relates to the gene therapy for treating inherited retinal degenerations (IRD) by expressing an opsin5 in retinal cells.
  • IRS inherited retinal degenerations
  • Inherited retinal degenerations are the leading cause of blindness, affecting 1 in 4,000 people worldwide ( ⁇ 1.75M in total) .
  • the photoreceptors e.g., rods and cones
  • Loss of photoreceptor cells and/or loss of photoreceptor cell function are the primary causes of reduced light sensitivity and blindness.
  • IRDs can result from different mutations in more than 300 genes and many identified forms have very low prevalence, which makes it unfeasible to develop gene specific treatments for all forms of IRDs.
  • retinal ganglion cells RRCs
  • opsins can enable the conversion of light into electro-chemical signals, thereby restoring the loss of vision caused by an IRD or other retinal degenerative diseases, in which the loss of photoreceptor cells and/or loss of photoreceptor cell function occur.
  • a new therapy for restoring vision such as a gene therapy for delivering the coding sequence of an opsin, particularly an opsin with having a higher sensitivity to light and expressing the same in retina to provide an improved therapeutic effect.
  • the present disclosure provides a polynucleotide construct comprising an expression cassette comprising a nucleotide sequence encoding a polypeptide comprising an opsin5 (OPN5) , operably linked to a promoter.
  • OPN5 opsin5
  • the OPN5 comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 2, 4, 6, 8 and 10.
  • the nucleotide sequence encodes a fusion polypeptide comprising the OPN5 and a fluorescent protein.
  • the fluorescent protein is a GFP or an RFP.
  • the promoter comprises a nucleotide sequence of SEQ ID NO: 19.
  • the expression cassette further comprises an enhancer comprising the nucleotide sequence of SEQ ID NO: 20 upstream of the promoter.
  • the expression cassette further comprises an intron comprising the nucleotide sequence of SEQ ID NO: 21 between the promoter and the nucleotide sequence.
  • the expression cassette further comprises a polyadenylation signal sequence comprising the nucleotide sequence of SEQ ID NO: 22 downstream of the nucleotide sequence.
  • the polynucleotide construct further comprises an expression cassette comprising a nucleotide sequence encoding a fluorescent protein.
  • the fluorescent protein is a GFP or an RFP.
  • the present disclosure provides a recombinant adeno-associated virus (rAAV) comprising a genome comprising the polynucleotide construct of the present disclosure.
  • rAAV recombinant adeno-associated virus
  • the genome further comprises a 5’-ITR of SEQ ID NO: 23 and a 3’-ITR of SEQ ID NO: 24.
  • the rAAV comprises a modified AAV2 capsid encoded by SEQ ID NO: 27.
  • the present disclosure provides a pharmaceutical composition comprising the rAAV of the present disclosure.
  • the present disclosure provides a method of treating vision loss caused by inherited retinal degenerations (IRD) , such as Stargardt’s Disease, Cone Rod Dystrophy, Retinitis Pigmentosa, Leber Congenital Amaurosis, Retinitis Pigmentosa (RP) , Best Juvenile Dystrophy, and Rod-Cone Dystrophies in a subject, comprising administering the rAAV or the pharmaceutical composition of the present disclosure to an eye of the subject.
  • IBD inherited retinal degenerations
  • the present disclosure also provides use of the rAAV or the pharmaceutical composition of the present disclosure to in the preparation of a medicament for treating vision loss caused by IRD, such as Stargardt’s Disease, Cone Rod Dystrophy, Retinitis Pigmentosa, Leber Congenital Amaurosis, Retinitis Pigmentosa (RP) , Best Juvenile Dystrophy, and Rod-Cone Dystrophies in a subject.
  • IRD vision loss caused by IRD
  • the present disclosure also provides the rAAV or the pharmaceutical composition of the present disclosure, for use in treating vision loss caused by IRD, such as Stargardt’s Disease, Cone Rod Dystrophy, Retinitis Pigmentosa, Leber Congenital Amaurosis, Retinitis Pigmentosa (RP) , Best Juvenile Dystrophy, and Rod-Cone Dystrophies in a subject.
  • vision loss caused by IRD such as Stargardt’s Disease, Cone Rod Dystrophy, Retinitis Pigmentosa, Leber Congenital Amaurosis, Retinitis Pigmentosa (RP) , Best Juvenile Dystrophy, and Rod-Cone Dystrophies in a subject.
  • the rAAV or the pharmaceutical composition is administered by intravitreal injection.
  • Fig. 1 shows the maps of the helper plasmid (Fig. 1A) , the packaging plasmid (Fig. 1B) and an exemplified transgene plasmid (Fig. 1C) .
  • Figs. 2 and 3 show the ERG detection in rd1 mice injected with rAAVs encoding opsin5 on Day 14 post-injection.
  • optical modulation refers to a technology that combines optics and genetics, based on a principle that photosensitive proteins (e.g., opsins) , which serve as the channels for cations or anions such as Cl-, Na+, H+, K+ through cellular membrane, are stimulated by light of specific wavelengths, resulting in changes in the membrane potential of the cell, thereby selectively exciting or inhibiting the cell.
  • Optogenetic modulation has a very high accuracy that it can achieve a modulation a single cell in the time at a sub-millisecond level.
  • Optogenetic modulation generally involves the construction of opsins, the expression of opsins, and the delivery of light. Due to the translucency of the eye structures, the delivery of light will not be a problem optogenetic modulation in eyes.
  • Opsin is a membrane protein with a molecular weight of approximately 30-50 kDa, which generally comprises one extracellular amino terminal, seven transmembrane regions, and one intracellular carboxyl terminal.
  • Opsins are members of G protein coupled receptor (GPCR) superfamily. Opsins can be found in animals and microorganisms. Non-animal opsins, such as those from archaea, bacteria, and fungi, have similar tertiary structures to animal opsins, but they are significantly different in amino acid sequences. In addition to native opsins, opsins include modified and artificial ones. Examples for opsins include but not limited to those listed in Table 1.
  • Adeno-associated virus is a member of Parvoviridae family. It is a simple single-stranded DNA virus, and requires a helper virus (such as adenovirus) for replication.
  • the genome of a wildtype AAV contains approximately 4.7 kilobases (kb) , comprising the cap and rep genes between two inverted terminal repeat (ITR) sequences, approximately 145 nucleotides in length, with interrupted palindromic sequences that can fold into hairpin structures that function as primers during initiation of DNA replication.
  • ITR inverted terminal repeat
  • the cap gene encodes the viral capsid protein
  • the rep gene is involved in the replication and integration of AAV.
  • AAV can infect a variety of cells, and the viral DNA can be integrated into human chromosome 19 in the presence of the rep product.
  • ITRs inverted terminal repeats
  • AAV viral cis-elements named due to their symmetry. These elements are essential for efficient multiplication of an AAV genome.
  • ITR refers to ITRs of known natural AAV serotypes, to chimeric ITRs formed by the fusion of ITR elements derived from different serotypes, and to functional variant thereof.
  • the production of a recombinant AAV particle may involve three plasmids, a transgene plasmid comprising an expression construct for expressing an exogenous polynucleotide, a packaging plasmid encoding the REP and/or CAP proteins, and a helper plasmid.
  • expression construct refers to a single-stranded or double-stranded polynucleotide, which is isolated from a naturally occurring gene or modified to contain a nucleic acid segment that does not naturally occur.
  • the expression construct may contain the control sequences required to express the coding sequence of the present disclosure.
  • polynucleotide usually refers to generally a nucleic acid molecule (e.g., 100 bases and up to 30 kilobases in length) and a sequence that is either complementary (antisense) or identical (sense) to the sequence of a messenger RNA (mRNA) or miRNA fragment or molecule.
  • mRNA messenger RNA
  • miRNA fragment or molecule usually refers to DNA or RNA molecules that are either transcribed or non-transcribed.
  • exogenous polynucleotide refers to a nucleotide sequence that does not originate from the host in which it is placed. It may be identical to the host’s DNA or heterologous. An example is a sequence of interest inserted into a vector. Such exogenous DNA sequences may be derived from a variety of sources including DNA, cDNA, synthetic DNA, and RNA. Exogenous polynucleotides also encompass DNA sequences that encode antisense oligonucleotides.
  • expression includes any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
  • control sequence includes all elements necessary or beneficial for the expression of the polynucleotide encoding the polypeptide of the present disclosure.
  • Each control sequence may be natural or foreign to the nucleotide sequence encoding the polypeptide, or natural or foreign to each other.
  • control sequences include, but are not limited to, leader sequence, polyadenylation sequence, propeptide sequence, promoter, enhancer, signal peptide sequence, and transcription terminator.
  • control sequences include a promoter and signals for the termination of transcription and translation.
  • control sequence may be a suitable promoter sequence, a nucleotide sequence recognized by the host cell to express the polynucleotide encoding the polypeptide of the present disclosure.
  • the promoter sequence contains a transcription control sequence that mediates the expression of the polypeptide.
  • the promoter may be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, for example, lac operon of E. coli.
  • the promoters also include mutant, modified and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides, which are homologous or heterologous to the host cell.
  • an intron can be included in the construct to improve the expression of the coding sequence.
  • the intron can be a native intron from a gene.
  • the intron can be an artificial one, such as a chimeric intron comprising a native intron and an exon, e.g., an exon from another gene.
  • operably linked refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence, whereby the control sequence directs the expression of the polypeptide coding sequence.
  • the polynucleotide encoding the opsin such as OPN5 of the present disclosure can be subjected to various manipulations to improve the expression of the polypeptide. Before the insertion thereof into a vector, manipulation of the polynucleotide according to the expression vector or the host, such as codon optimization, is desirable or necessary.
  • recombinant refers to nucleic acids, vectors, polypeptides, or proteins that have been generated using DNA recombination (cloning) methods and are distinguishable from native or wild-type nucleic acids, vectors, polypeptides, or proteins.
  • polypeptide and “protein” are used interchangeably herein and refer to a polymer of amino acids and includes full-length proteins and fragments thereof.
  • the term “host cell” refers to, for example microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of rAAV vectors.
  • the term includes the progeny of the original cell which has been transduced.
  • a “host cell” as used herein generally refers to a cell which has been transduced with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to natural, accidental, or deliberate mutation.
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce toxicity or an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • subject includes, but is not limited to, humans, nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like.
  • farm animals such as cattle, sheep, pigs, goats and horses
  • domestic mammals such as dogs and cats
  • laboratory animals including rodents such as mice, rats and guinea pigs, and the like.
  • rodents such as mice, rats and guinea pigs, and the like.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
  • the present disclosure provides a polynucleotide construct comprising an expression cassette comprising a nucleotide sequence encoding a polypeptide comprising an opsin5 (OPN5) , operably linked to a promoter.
  • OPN5 opsin5
  • the nucleotide sequence is codon optimized to improve the expression of the fusion polypeptide in mammal cells, e.g., human cells.
  • the OPN5 is sensitive to blue light, e.g., the OPN5 has a peak activation with a wavelength in the range of 400-500nm, such as 400nm, 405nm, 410nm, 415nm, 420nm, 425nm, 430nm, 435nm, 440nm, 445nm, 450nm, 455nm, 460nm, 465nm, 470nm, 475nm, 480nm, 485nm, 490nm, 495nm or 500nm, and the first wavelength is 400nm, 405nm, 410nm, 415nm, 420nm, 425nm, 430nm, 435nm, 440nm, 445nm, 450nm, 455nm, 460nm, 465nm, 470nm, 475nm, 480nm, 485nm, 490nm, 495nm or 500nm, and
  • the OPN5 is an OPN5 from Accipiter strictis (AGOPN5) , Alligator mississippiensis (AMOPN5) , Gallus gallus (cOPN5/GGOPN5) , Pipistrellus kuhlii (PKOPN5) , or Serinus canaria (SCOPN5) .
  • the OPN comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8 and 10, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to one of SEQ ID NOs: 2, 4, 6, 8 and 10.
  • the OPN5 is responsive to red light, e.g., the OPN5 has a peak activation with a wavelength in the range of 550-650nm, such as 550nm, 555nm, 560nm, 565nm, 570nm, 575nm, 580nm, 585nm, 590nm, 595nm, 600nm, 605nm, 610nm, 615nm, 620nm, 625nm, 630nm, 635nm, 640nm, 645nm or 650nm.
  • 550-650nm such as 550nm, 555nm, 560nm, 565nm, 570nm, 575nm, 580nm, 585nm, 590nm, 595nm, 600nm, 605nm, 610nm, 615nm, 620nm, 625nm, 630nm, 635nm, 640nm, 645n
  • the OPN5 is AMOPN5, PKOPN5 or SCOPN5.
  • the OPN comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8 and 10, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to one of SEQ ID NOs: 4, 8 and 10.
  • the expression cassette comprising a nucleotide sequence comprising SEQ ID NOs: 1, 3, 5, 7 and 9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to one of SEQ ID NOs: 1, 3, 5, 7 and 9.
  • the expression cassette comprising a nucleotide sequence comprising SEQ ID NOs: 3, 7 and 9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to one of SEQ ID NOs: 3, 7 and 9.
  • the fluorescent protein can be co-expressed with the OPN5 in a single expression cassette, or in separate cassettes.
  • the polynucleotide construct comprises an expression cassette comprising a nucleotide sequence encoding the OPN5 and the fluorescent protein.
  • the OPN5 and the fluorescent protein are encoded as a fusion polypeptide, in which the OPN5 is linked to the fluorescent protein by a linker.
  • the linker comprises a GS linker such as GSG.
  • the linker further comprises a cleavable site, such as Furin site (e.g., SEQ ID NO: 14) and/or a 2A peptide (e.g., T2A of SEQ ID NO: 16) .
  • the linker comprises SEQ ID NO: 14, GSG, and SEQ ID NO: 16 from N terminus to C terminus.
  • the polynucleotide construct comprises an expression cassette comprising a nucleotide sequence encoding the OPN5, and an expression cassette comprising a nucleotide sequence encoding the fluorescent protein.
  • the fluorescent protein is a GFP or an RFP.
  • the fluorescent protein is an EGFP of SEQ ID NO: 18.
  • the promoter comprises a nucleotide sequence of SEQ ID NO: 19 or a nucleotide sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to SEQ ID NO: 19.
  • the expression cassette further comprises an enhancer comprising the nucleotide sequence of SEQ ID NO: 20 or a nucleotide sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to SEQ ID NO: 20 upstream of the promoter.
  • the expression cassette further comprises an intron comprising the nucleotide sequence of SEQ ID NO: 21 or a nucleotide sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to SEQ ID NO: 21 between the promoter and the nucleotide sequence.
  • the expression cassette further comprises a polyadenylation signal sequence comprising the nucleotide sequence of SEQ ID NO: 22 downstream of the nucleotide sequence or a nucleotide sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to SEQ ID NO: 22.
  • the polynucleotide construct comprises, from 5’ to 3’ end, SEQ ID NO: 20, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 1, and SEQ ID NO: 22.
  • the polynucleotide construct comprises, from 5’ to 3’ end, SEQ ID NO: 20, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 3, and SEQ ID NO: 22.
  • the polynucleotide construct comprises, from 5’ to 3’ end, SEQ ID NO: 20, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 5, and SEQ ID NO: 22.
  • the polynucleotide construct comprises, from 5’ to 3’ end, SEQ ID NO: 20, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 7, and SEQ ID NO: 22.
  • the polynucleotide construct comprises, from 5’ to 3’ end, SEQ ID NO: 20, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 9, and SEQ ID NO: 22.
  • the polynucleotide construct comprises, from 5’ to 3’ end, SEQ ID NO: 20, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 1, SEQ ID NO: 13, GGATCCGGC, SEQ IDNO: 15, SEQ ID NO: 17, and SEQ ID NO: 22.
  • the polynucleotide construct comprises, from 5’ to 3’ end, SEQ ID NO: 20, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 3, SEQ ID NO: 13, GGATCCGGC, SEQ IDNO: 15, SEQ ID NO: 17, and SEQ ID NO: 22.
  • the polynucleotide construct comprises, from 5’ to 3’ end, SEQ ID NO: 20, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 5, SEQ ID NO: 13, GGATCCGGC, SEQ IDNO: 15, SEQ ID NO: 17, and SEQ ID NO: 22.
  • the polynucleotide construct comprises, from 5’ to 3’ end, SEQ ID NO: 20, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 7, SEQ ID NO: 13, GGATCCGGC, SEQ IDNO: 15, SEQ ID NO: 17, and SEQ ID NO: 22.
  • the polynucleotide construct comprises, from 5’ to 3’ end, SEQ ID NO: 20, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 9, SEQ ID NO: 13, GGATCCGGC, SEQ IDNO: 15, SEQ ID NO: 17, and SEQ ID NO: 22.
  • a vector comprising the polynucleotide construct of the present disclosure, and a host cell comprising the polynucleotide construct or the vector of the present disclosure.
  • the vector can be a transgene plasmid for preparing an rAAV. Therefore, in some embodiment, the vector comprises the polynucleotide construct and AAV ITRs flanking the polynucleotide construct. In some embodiments, the vector comprises AAV2 ITRs. In some embodiments, the 5’ ITR comprises a nucleotide sequence of SEQ ID NO: 23, or a nucleotide sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to SEQ ID NO: 23.
  • the 3’ ITR comprises a nucleotide sequence of SEQ ID NO: 24, or a nucleotide sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to SEQ ID NO: 24.
  • the present disclosure provides an rAAV for delivering and expressing the polynucleotide construct encoding a OPN5 in retinal cells, such as RGCs, thereby restoring the loss of vision due to the degeneration of photoreceptors such as rods and cones.
  • the rAAV comprises a genome comprising a polynucleotide construct comprising an expression cassette comprising a nucleotide sequence encoding a polypeptide comprising an opsin5 (OPN5) , operably linked to a promoter.
  • OPN5 opsin5
  • the nucleotide sequence is codon optimized to improve the expression of the fusion polypeptide in mammal cells, e.g., human cells.
  • the OPN5 is sensitive to blue light, e.g., the OPN5 has a peak activation with a wavelength in the range of 400-500nm, such as 400nm, 405nm, 410nm, 415nm, 420nm, 425nm, 430nm, 435nm, 440nm, 445nm, 450nm, 455nm, 460nm, 465nm, 470nm, 475nm, 480nm, 485nm, 490nm, 495nm or 500nm, and the first wavelength is 400nm, 405nm, 410nm, 415nm, 420nm, 425nm, 430nm, 435nm, 440nm, 445nm, 450nm, 455nm, 460nm, 465nm, 470nm, 475nm, 480nm, 485nm, 490nm, 495nm or 500nm, and
  • the OPN5 is an OPN5 from Accipiter strictis (AGOPN5) , Alligator mississippiensis (AMOPN5) , Gallus gallus (cOPN5/GGOPN5) , Pipistrellus kuhlii (PKOPN5) , or Serinus canaria (SCOPN5) .
  • the OPN comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8 and 10, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to one of SEQ ID NOs: 2, 4, 6, 8 and 10.
  • the OPN5 is responsive to red light, e.g., the OPN5 has a peak activation with a wavelength in the range of 550-650nm, such as 550nm, 555nm, 560nm, 565nm, 570nm, 575nm, 580nm, 585nm, 590nm, 595nm, 600nm, 605nm, 610nm, 615nm, 620nm, 625nm, 630nm, 635nm, 640nm, 645nm or 650nm.
  • 550-650nm such as 550nm, 555nm, 560nm, 565nm, 570nm, 575nm, 580nm, 585nm, 590nm, 595nm, 600nm, 605nm, 610nm, 615nm, 620nm, 625nm, 630nm, 635nm, 640nm, 645n
  • the OPN5 is AMOPN5, PKOPN5 or SCOPN5.
  • the OPN comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8 and 10, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to one of SEQ ID NOs: 4, 8 and 10.
  • the expression cassette comprising a nucleotide sequence comprising SEQ ID NOs: 1, 3, 5, 7 and 9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to one of SEQ ID NOs: 1, 3, 5, 7 and 9.
  • the expression cassette comprising a nucleotide sequence comprising SEQ ID NOs: 3, 7 and 9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to one of SEQ ID NOs: 3, 7 and 9.
  • the fluorescent protein can be co-expressed with the OPN5 in a single expression cassette, or in separate cassettes.
  • the polynucleotide construct comprises an expression cassette comprising a nucleotide sequence encoding the OPN5 and the fluorescent protein.
  • the OPN5 and the fluorescent protein are encoded as a fusion polypeptide, in which the OPN5 is linked to the fluorescent protein by a linker.
  • the linker comprises a GS linker such as GSG.
  • the linker further comprises a cleavable site, such as Furin site (e.g., SEQ ID NO: 14) and/or a 2A peptide (e.g., T2A of SEQ ID NO: 16) .
  • the linker comprises SEQ ID NO: 14, GSG, and SEQ ID NO: 16 from N terminus to C terminus.
  • the polynucleotide construct comprises an expression cassette comprising a nucleotide sequence encoding the OPN5, and an expression cassette comprising a nucleotide sequence encoding the fluorescent protein.
  • the fluorescent protein is a GFP or an RFP.
  • the fluorescent protein is an EGFP of SEQ ID NO: 18.
  • the promoter comprises a nucleotide sequence of SEQ ID NO: 19 or a nucleotide sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to SEQ ID NO: 19.
  • the expression cassette further comprises an enhancer comprising the nucleotide sequence of SEQ ID NO: 20 or a nucleotide sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to SEQ ID NO: 20 upstream of the promoter.
  • the expression cassette further comprises an intron comprising the nucleotide sequence of SEQ ID NO: 21 or a nucleotide sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to SEQ ID NO: 21 between the promoter and the nucleotide sequence.
  • the expression cassette further comprises a polyadenylation signal sequence comprising the nucleotide sequence of SEQ ID NO: 22 downstream of the nucleotide sequence or a nucleotide sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to SEQ ID NO: 22.
  • the polynucleotide construct comprises, from 5’ to 3’ end, SEQ ID NO: 20, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 1, and SEQ ID NO: 22.
  • the polynucleotide construct comprises, from 5’ to 3’ end, SEQ ID NO: 20, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 3, and SEQ ID NO: 22.
  • the polynucleotide construct comprises, from 5’ to 3’ end, SEQ ID NO: 20, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 5, and SEQ ID NO: 22.
  • the polynucleotide construct comprises, from 5’ to 3’ end, SEQ ID NO: 20, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 7, and SEQ ID NO: 22.
  • the polynucleotide construct comprises, from 5’ to 3’ end, SEQ ID NO: 20, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 9, and SEQ ID NO: 22.
  • the polynucleotide construct comprises, from 5’ to 3’ end, SEQ ID NO: 20, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 1, SEQ ID NO: 13, GGATCCGGC, SEQ IDNO: 15, SEQ ID NO: 17, and SEQ ID NO: 22.
  • the polynucleotide construct comprises, from 5’ to 3’ end, SEQ ID NO: 20, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 3, SEQ ID NO: 13, GGATCCGGC, SEQ IDNO: 15, SEQ ID NO: 17, and SEQ ID NO: 22.
  • the polynucleotide construct comprises, from 5’ to 3’ end, SEQ ID NO: 20, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 5, SEQ ID NO: 13, GGATCCGGC, SEQ IDNO: 15, SEQ ID NO: 17, and SEQ ID NO: 22.
  • the polynucleotide construct comprises, from 5’ to 3’ end, SEQ ID NO: 20, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 7, SEQ ID NO: 13, GGATCCGGC, SEQ IDNO: 15, SEQ ID NO: 17, and SEQ ID NO: 22.
  • the polynucleotide construct comprises, from 5’ to 3’ end, SEQ ID NO: 20, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 9, SEQ ID NO: 13, GGATCCGGC, SEQ IDNO: 15, SEQ ID NO: 17, and SEQ ID NO: 22.
  • the genome further comprises AAV ITRs flanking the polynucleotide construct.
  • the vector comprises AAV2 ITRs.
  • the 5’ ITR comprises a nucleotide sequence of SEQ ID NO: 23, or a nucleotide sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to SEQ ID NO: 23.
  • the 3’ ITR comprises a nucleotide sequence of SEQ ID NO:24, or a nucleotide sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to SEQ ID NO: 24.
  • the rAAV of the present disclosure can be selected from human serotype 1 AAV (hAAV1) , hAAV2, hAAV3, hAAV4, hAAV5, hAAV6, hAAV7, hAAV8, hAAV9, hAAV10, and hAAV11.26.
  • the rAAV is hAAV2.
  • a number of modified AAV capsids derived from hAAV2 have been reported, which improve gene therapy for ocular diseases.
  • the rAAV comprises a modified AAV2 capsid, such as those described in WO2012145601, WO2016134375, WO2018022905 and WO2023155828, preferably a capsid encoded by SEQ ID NO: 27.
  • rAAV It is known in the art to package rAAV with a system comprising three plasmids, including i) a transgene plasmid comprising the genome of the rAAV encoding a desired gene product, ii) a packaging plasmid encoding the REP and/or CAP proteins, and iii) a helper plasmid in a host cell (see, e.g., Crosson SM et al., Helper-free Production of Laboratory Grade AAV and Purification by Iodixanol Density Gradient Centrifugation. Mol Ther Methods Clin Dev. 2018; 10: 1-7) .
  • a method of producing rAAV is also described in, for example, U.S. Patent Publication No. 2005/0053922 and U.S. Patent Publication No. 2009/0202490.
  • polynucleotide construct or the vector of the present disclosure can be introduced into the host cell stably or transiently using defined techniques including, but not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection, and the like.
  • the subject nucleic acid will typically be operably linked to a selectable marker, such as neomycin resistance gene and the like.
  • the host cell is a variety of cells, such as mammalian cells, including, for example, murine cells and primate cells (e.g., human cells) .
  • Suitable mammalian cells include, but are not limited to, primary cells and cell lines, wherein suitable cell lines include, but are not limited to, 293 cells, COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, C3H10T1/2 fibroblasts, CHO cells, etc.
  • suitable host cells include, for example, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2) , CHO cells (e.g., ATCC No.
  • CRL9618, CCL61, CRL9096 293 cells (e.g., ATCC No. CRL-1573) , Vero cells, NIH3T3 cells (eg, ATCC No. CRL-1658) , Huh-7 cells, BHK cells (e.g., ATCC No. CCL10) , PC12 cells (ATCC No. CRL1721) COS cells, COS-7 cells (ATCC No. CRL1651) , RAT1 cells, mouse L cells (ATCC No. CCLI. 3) , human embryonic kidney (HEK) cells (ATCC No. CRL1573) , HLHepG2 cells, and the like.
  • ATCC No. CRL-1573 Vero cells
  • NIH3T3 cells eg, ATCC No. CRL-1658
  • Huh-7 cells BHK cells (e.g., ATCC No. CCL10)
  • PC12 cells ATCC No. CRL1721) COS cells
  • COS-7 cells ATCC No. CRL1651
  • Bacterial cells such as Sf9 cells, which produce AAV, can also be used to prepare the host cell of the present disclosure (see, for example, U.S. Patent No. 7,271,002; U.S. Patent Publication No. 12/297,958) .
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising: a) the rAAV of the present disclosure; and b) a pharmaceutically acceptable carrier, diluent, excipient or buffer.
  • a pharmaceutically acceptable carrier, diluent, excipient or buffer is suitable for use in humans.
  • excipients include any agent that can be administered without abnormal toxicity.
  • Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, and ethanol. Included therein may be pharmaceutically acceptable salts such as mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and salts of organic acids such as acetates, propionates, malonates, Benzoate and the like.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may be present in such vehicles.
  • the present disclosure provides a method of treating an ocular disease with loss of photoreceptor, the method comprising expressing an OPN5 or a fusion polypeptide comprising OPN5 in retinal cells, such as RGCs.
  • the nucleotide sequence is codon optimized to improve the expression of the fusion polypeptide in mammal cells, e.g., human cells.
  • the OPN5 is sensitive to blue light, e.g., the OPN5 has a peak activation with a wavelength in the range of 400-500nm, such as 400nm, 405nm, 410nm, 415nm, 420nm, 425nm, 430nm, 435nm, 440nm, 445nm, 450nm, 455nm, 460nm, 465nm, 470nm, 475nm, 480nm, 485nm, 490nm, 495nm or 500nm, and the first wavelength is 400nm, 405nm, 410nm, 415nm, 420nm, 425nm, 430nm, 435nm, 440nm, 445nm, 450nm, 455nm, 460nm, 465nm, 470nm, 475nm, 480nm, 485nm, 490nm, 495nm or 500nm, and
  • the OPN5 is an OPN5 from Accipiter strictis (AGOPN5) , Alligator mississippiensis (AMOPN5) , Gallus gallus (cOPN5/GGOPN5) , Pipistrellus kuhlii (PKOPN5) , or Serinus canaria (SCOPN5) .
  • the OPN comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8 and 10, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to one of SEQ ID NOs: 2, 4, 6, 8 and 10.
  • the OPN5 is responsive to red light, e.g., the OPN5 has a peak activation with a wavelength in the range of 550-650nm, such as 550nm, 555nm, 560nm, 565nm, 570nm, 575nm, 580nm, 585nm, 590nm, 595nm, 600nm, 605nm, 610nm, 615nm, 620nm, 625nm, 630nm, 635nm, 640nm, 645nm or 650nm.
  • 550-650nm such as 550nm, 555nm, 560nm, 565nm, 570nm, 575nm, 580nm, 585nm, 590nm, 595nm, 600nm, 605nm, 610nm, 615nm, 620nm, 625nm, 630nm, 635nm, 640nm, 645n
  • the OPN5 is AMOPN5, PKOPN5 or SCOPN5.
  • the OPN comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8 and 10, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to one of SEQ ID NOs: 4, 8 and 10.
  • the expression cassette comprising a nucleotide sequence comprising SEQ ID NOs: 1, 3, 5, 7 and 9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to one of SEQ ID NOs: 1, 3, 5, 7 and 9.
  • the expression cassette comprising a nucleotide sequence comprising SEQ ID NOs: 3, 7 and 9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to one of SEQ ID NOs: 3, 7 and 9.
  • the method of expressing a polypeptide of interest in a cell includes, but is not limited to, delivering a polynucleotide encoding the polypeptide to the retinal cell, such as RGCs.
  • the polynucleotide includes, but is not limited to, a plasmid, a viral vector, and an mRNA.
  • the viral vector is an rAAV vector.
  • the method comprises administering to a subject in need thereof an effective amount of the rAAV or the pharmaceutical composition of the present disclosure.
  • the rAAV or the pharmaceutical composition is administered by intraocular injection or by intravitreal injection.
  • the rAAV or the pharmaceutical composition is administered by a single-dose or a multiple-dose (e.g., 2, 3, 4 or more doses) scheme.
  • the rAAV or the pharmaceutical composition can be administered at different intervals, such as daily, weekly, monthly, yearly, to achieve a desired level of gene expression.
  • an OPN5 polypeptide or a polynucleotide encoding an OPN5 polypeptide for use in the treatment of an ocular disease with loss of photoreceptor.
  • the nucleotide sequence is codon optimized to improve the expression of the fusion polypeptide in mammal cells, e.g., human cells.
  • the OPN5 is sensitive to blue light, e.g., the OPN5 has a peak activation with a wavelength in the range of 400-500nm, such as 400nm, 405nm, 410nm, 415nm, 420nm, 425nm, 430nm, 435nm, 440nm, 445nm, 450nm, 455nm, 460nm, 465nm, 470nm, 475nm, 480nm, 485nm, 490nm, 495nm or 500nm, and the first wavelength is 400nm, 405nm, 410nm, 415nm, 420nm, 425nm, 430nm, 435nm, 440nm, 445nm, 450nm, 455nm, 460nm, 465nm, 470nm, 475nm, 480nm, 485nm, 490nm, 495nm or 500nm, and
  • the OPN5 is an OPN5 from Accipiter strictis (AGOPN5) , Alligator mississippiensis (AMOPN5) , Gallus gallus (cOPN5/GGOPN5) , Pipistrellus kuhlii (PKOPN5) , or Serinus canaria (SCOPN5) .
  • the OPN comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8 and 10, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to one of SEQ ID NOs: 2, 4, 6, 8 and 10.
  • the OPN5 is responsive to red light, e.g., the OPN5 has a peak activation with a wavelength in the range of 550-650nm, such as 550nm, 555nm, 560nm, 565nm, 570nm, 575nm, 580nm, 585nm, 590nm, 595nm, 600nm, 605nm, 610nm, 615nm, 620nm, 625nm, 630nm, 635nm, 640nm, 645nm or 650nm.
  • 550-650nm such as 550nm, 555nm, 560nm, 565nm, 570nm, 575nm, 580nm, 585nm, 590nm, 595nm, 600nm, 605nm, 610nm, 615nm, 620nm, 625nm, 630nm, 635nm, 640nm, 645n
  • the OPN5 is AMOPN5, PKOPN5 or SCOPN5.
  • the OPN comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8 and 10, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to one of SEQ ID NOs: 4, 8 and 10.
  • the expression cassette comprising a nucleotide sequence comprising SEQ ID NOs: 1, 3, 5, 7 and 9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to one of SEQ ID NOs: 1, 3, 5, 7 and 9.
  • the expression cassette comprising a nucleotide sequence comprising SEQ ID NOs: 3, 7 and 9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to one of SEQ ID NOs: 3, 7 and 9.
  • the rAAV or the pharmaceutical composition of the present disclosure for use in the treatment of an ocular disease with loss of photoreceptor.
  • the rAAV or the pharmaceutical composition is administered by intraocular injection or by intravitreal injection.
  • the rAAV or the pharmaceutical composition is administered by a single-dose or a multiple-dose (e.g., 2, 3, 4 or more doses) scheme.
  • the rAAV or the pharmaceutical composition can be administered at different intervals, such as daily, weekly, monthly, yearly, to achieve a desired level of gene expression.
  • an OPN5 polypeptide or a polynucleotide encoding an OPN5 polypeptide in the preparation of a medicament for treating an ocular disease with loss of photoreceptor.
  • the nucleotide sequence is codon optimized to improve the expression of the fusion polypeptide in mammal cells, e.g., human cells.
  • the OPN5 is sensitive to blue light, e.g., the OPN5 has a peak activation with a wavelength in the range of 400-500nm, such as 400nm, 405nm, 410nm, 415nm, 420nm, 425nm, 430nm, 435nm, 440nm, 445nm, 450nm, 455nm, 460nm, 465nm, 470nm, 475nm, 480nm, 485nm, 490nm, 495nm or 500nm, and the first wavelength is 400nm, 405nm, 410nm, 415nm, 420nm, 425nm, 430nm, 435nm, 440nm, 445nm, 450nm, 455nm, 460nm, 465nm, 470nm, 475nm, 480nm, 485nm, 490nm, 495nm or 500nm, and
  • the OPN5 is an OPN5 from Accipiter strictis (AGOPN5) , Alligator mississippiensis (AMOPN5) , Gallus gallus (cOPN5/GGOPN5) , Pipistrellus kuhlii (PKOPN5) , or Serinus canaria (SCOPN5) .
  • the OPN comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8 and 10, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to one of SEQ ID NOs: 2, 4, 6, 8 and 10.
  • the OPN5 is responsive to red light, e.g., the OPN5 has a peak activation with a wavelength in the range of 550-650nm, such as 550nm, 555nm, 560nm, 565nm, 570nm, 575nm, 580nm, 585nm, 590nm, 595nm, 600nm, 605nm, 610nm, 615nm, 620nm, 625nm, 630nm, 635nm, 640nm, 645nm or 650nm.
  • 550-650nm such as 550nm, 555nm, 560nm, 565nm, 570nm, 575nm, 580nm, 585nm, 590nm, 595nm, 600nm, 605nm, 610nm, 615nm, 620nm, 625nm, 630nm, 635nm, 640nm, 645n
  • the OPN5 is AMOPN5, PKOPN5 or SCOPN5.
  • the OPN comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8 and 10, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to one of SEQ ID NOs: 4, 8 and 10.
  • the expression cassette comprising a nucleotide sequence comprising SEQ ID NOs: 1, 3, 5, 7 and 9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to one of SEQ ID NOs: 1, 3, 5, 7 and 9.
  • the expression cassette comprising a nucleotide sequence comprising SEQ ID NOs: 3, 7 and 9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more identical to one of SEQ ID NOs: 3, 7 and 9.
  • the present disclosure provides use of the rAAV or the pharmaceutical composition of the present disclosure in the preparation of a medicament for treating an ocular disease with loss of photoreceptor in a subject in need thereof.
  • the OPN5s, the polynucleotide encoding the OPN5, the rAAV or the pharmaceutical composition of the present disclosure can be used to treat vision loss due to any degenerative retinal disease, which includes, but is not limited to, inherited retinal degenerations (IRDs) , such as Stargardt’s Disease, Cone Rod Dystrophy, Retinitis Pigmentosa, Leber Congenital Amaurosis (LCA) , Retinitis Pigmentosa (RP) , Best Juvenile Dystrophy, and Rod-Cone Dystrophies.
  • ITDs inherited retinal degenerations
  • Example 1 Construction of transgene plasmids and rAAVs expressing an OPN5s or a benchmark
  • This Example was carried out to detect the light sensitivity of OPN5s.
  • Transgene plasmids for expressing an OPN5 were constructed by assembling the elements (a CMV IE enhancer of SEQ ID NO: 20, a CBA promoter of SEQ ID NO: 19, a chimeric intron of SEQ ID NO: 21, a coding sequence (consisting of OPN5, Furin, GSG and T2A) , a hGH polyA signal of SEQ ID NO: 22, and ITRs of SEQ ID NOs: 23 and 24) using Gibson assembly methodology (NEBuilder HiFi DNA Assembly Master Mix, NEB, Catalog E2621) . As shown in Fig.
  • OPN5 is a nucleotide sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9 (encoding AGOPN5, AMOPN5, cOPN5/GGOPN5, PKOPN5, and SCOPN5, respectively)
  • EGFP is a nucleotide sequence of SEQ ID NO: 17
  • Furin is a nucleotide sequence of SEQ ID NO: 13
  • T2A is a nucleotide sequence of SEQ ID NO: 12 and the GSG is encoded by GGATCCGGC.
  • the rAAV genomes (5’ ITR to 3’ ITR) encoding the AGOPN5, AMOPN5, cOPN5/GGOPN5, PKOPN5, and SCOPN5, respectively were shown in SEQ ID NOs: 29-33 as examples.
  • transgene plasmids were constructed as described above with the coding sequence replaced by SEQ ID NO: 25 (referred to as 2838GS hereinafter) or 26 (referred to as 3210GS or SKG1101 hereinafter) , which encode two fusion polypeptides designed by the inventor, comprising an opsin sensitive to blue light, an opsin sensitive to red light, and a fluorescent protein.
  • 2838GS and 3210GS had shown improved restoration of vision in rd1 mice as compared to the fusion polypeptide of opsins disclosed in the prior art (e.g., WO 2018/106369) .
  • rAAVs were prepared with a method similar to that described in Crosson SM et al. 2018. Briefly, 3E6 cells/ml 293VPC cells (Thermo, Catalog A35347) in serum free virus production medium OPM-293 CD05 (Shanghai OPM Biosciences Co. Ltd. Catalog: 81075-001) were triple transfected, using polyethylenimine, with the helper plasmid, the packaging plasmid encoding rep/cap, and the transgene plasmid below:
  • helper containing the Ad E2A, E4, and VA RNA helper genes as described in Crosson Sm et al., the map thereof is shown in Fig. 1A) ;
  • FIG. 1B the map thereof is shown in Fig. 1B, in which “AAV2 Cap” is the nucleotide sequence encoding a modified AAV2 capsid polypeptide (SEQ ID NO: 27) , and “AAV2 Rep” is the nucleotide sequence encoding an AAV2 Rep polypeptide (SEQ ID NO: 28) ;
  • the rAAVs were tested for titers by ddPCR, which were all at the level of 10 13 viral genomes (vg) /mL.
  • ddPCR was carried out with Bio-Rad’s QXDx AutoDG ddPCR System and QXDx Universal Kit for AutoDG ddPCR System according to the manufacturer’s instructions.
  • the primers and probe for the ddPCR are as follows:
  • transgene plasmids and rAAVs were referred to as coding sequence for the opsin or fusion polypeptide of opsin expressed thereby.
  • This Example was carried out to detect whether the OPN5s can provide a superior optogenetic modulation for vision restoration.
  • the rAAVs prepared in Example 1 were administered to rd1 mice (Jackson Laboratory, 004766, 6-8 week-old) by an intravitreal (IVT) injection at a dose of 7.5E9 vg/eye (12 eyes/group with wildtype mice, and rd1 mice injected with PBS as positive control and negative control) .
  • IVT intravitreal
  • Electroretinogram (ERG) detection was conducted 14 days after the injection.
  • mice were anesthetized and pupils were dilated with 1%Tropicamide and 1%Atropine.
  • ERGs RetiMINER-C, IRC
  • IRC Electroretinogram
  • mice were dark-adapted overnight and stimulated with flashes (white light, blue light (470nm) , green light (520nm) and red light (590nm) ) of steadily increasing light intensity (0.1, 1, and 10 log cds/m 2 ) .
  • flashes white light, blue light (470nm) , green light (520nm) and red light (590nm) ) of steadily increasing light intensity (0.1, 1, and 10 log cds/m 2 ) .
  • the data were analyzed via RetiMINER4.0.
  • mice injected with rAAVs expressing OPN5s showed improved efficacy than 2838GS and 3210GS in response to blue light, while AMOPN5, SCOPN5, and PKOPN5 even showed improved efficacy than 2838GS and 3210GS in response to red light and white light.

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Abstract

La présente invention concerne une construction polynucléotidique comprenant une cassette d'expression comportant une séquence nucléotidique codant pour un polypeptide comprenant une opsine 5 (OPN5), lié de manière fonctionnelle à un promoteur. La présente divulgation concerne également un rAAV pour exprimer l'OPN5 dans des cellules rétiniennes, telles que des RGC.
PCT/CN2025/088056 2024-04-10 2025-04-09 Modulation optogénétique pour restauration de la vision Pending WO2025214408A1 (fr)

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