Vectors encoding rod-derived cone survival factors and human IgK signaling sequences.

Abstract

This invention relates to nucleic acids that encode and express full-length rod-derived cone survival factor (RdCVF) and human IgK signaling sequences, as well as viral vectors containing these nucleic acids. The invention also relates to compositions and pharmaceutical preparations containing these nucleic acids or vectors, methods for producing or secreting full-length RdCVF and human IgK signaling sequences, and therapeutic methods.

Description

[Background technology]

[0001] Rod-derived cone survival factor (RdCVF) is a thioredoxin-like protein specifically expressed by rod photoreceptor cells in the retina (Leveillard et al. (2004) Nature Genetics 36:755-759 and supplemental information). Two distinct RdCVF genes are found in humans, called RdCVF1 and RdCVF2. Both RdCVF genes encode two products via alternative splicing: a full-length protein and a protein with its C-terminus post-transcriptionally shortened, known as RdCVF-long and RdCVF-short, respectively.

[0002] RdCVF1-short is described as a secreted trophoblast that promotes pyramidal survival, while RdCVF1-long is described as a redox-active enzyme that interacts with intracellular proteins (Leveillard et al. (2010) Sci Transl Med. 2(26):26ps16). For example, tau is described as a binding partner of RdCVF1-L, and tau is found only intracellularly (Fridlich et al. (2009) Molecular & Cellular Proteomics 8(6):1206-18).

[0003] Any reference or discussion of prior art in this specification should not be construed as an admission that such art is prior art to the present invention. [Overview of the project]

[0004] In embodiments, the present disclosure provides a nucleic acid encoding a full rod-derived cone survival factor ("RdCVF-long" or "RdCVFL") protein and a nucleotide sequence encoding a human immunoglobulin kappa chain (IgK) signal sequence, wherein the human IgK signal sequence comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 7.

[0005] In embodiments, the Disclosure provides nucleic acids disclosed herein, wherein the RdCVFL protein is either the RdCVF1L protein or the RdCVF2L protein.

[0006] In an embodiment, the present disclosure provides a nucleic acid in which the RdCVFL protein is the RdCVF1L protein.

[0007] In embodiments, the Disclosure provides nucleic acids disclosed herein, wherein the RdCVFL protein is a human RdCVFL protein.

[0008] In embodiments, the Disclosure provides nucleic acids disclosed herein, wherein the nucleotide sequence encoding the RdCVFL protein comprises a re-encoded nucleotide sequence.

[0009] In embodiments, the Disclosure provides nucleic acids disclosed herein in which the recoded nucleotide sequence lacks an start methionine codon.

[0010] In embodiments, the Disclosure provides nucleic acids disclosed herein, wherein the human IgK signaling sequence is N-terminus of the RdCVFL protein.

[0011] In embodiments, the Disclosure provides nucleic acids disclosed herein in which a nucleotide sequence encoding a human IgK signaling sequence is operably linked to a nucleotide sequence encoding a human RdCVFL.

[0012] In embodiments, the Disclosure provides nucleic acids disclosed herein, wherein the RdCVFL protein and human IgK signaling sequence include an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 3 or SEQ ID NO: 15.

[0013] In embodiments, the Disclosure provides nucleic acids disclosed herein, wherein the RdCVFL protein and human IgK signaling sequence include an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 3.

[0014] In embodiments, the Disclosure provides nucleic acids disclosed herein, wherein the recoded nucleotide sequence has at least 40% of its codons recoded.

[0015] In embodiments, the Disclosure provides nucleic acids disclosed herein, wherein the recoded nucleotide sequence differs by at least 15% of the nucleotides compared to the corresponding native nucleotide sequence.

[0016] In embodiments, the Disclosure provides nucleic acids disclosed herein, wherein the recoded nucleotide sequence is less than 90% identical to the corresponding native nucleotide sequence.

[0017] In embodiments, the Disclosure provides nucleic acids disclosed herein, wherein the nucleotide sequence or recoded nucleotide sequence has one or more characteristics selected from the following: absence of a procaria inhibitory motif, absence of a consensus splice donor site, absence of a cryptic splice donor site, and a GC content of 60-65%.

[0018] In embodiments, the Disclosure provides nucleic acids disclosed herein, in which a promoter sequence is operably linked to a human IgK signal sequence and a nucleotide sequence encoding a human RdCVFL protein.

[0019] In embodiments, the disclosure includes promoters such as the phage-lambda (PL) promoter; the SV40 initial promoter; the herpes simplex virus (HSV) promoter; the cytomegalovirus (CMV) promoter; hybrid promoters including the CMV enhancer and chicken beta-actin promoter; the tetracycline regulatory transactivator-responsive promoter (tet) system; long-terminal repeat (LTR) promoters such as MoMLV LTR, BIV LTR, or HIV LTR; the Moloney mouse sarcoma virus U3 region promoter; the granzyme A promoter; the regulatory sequence of the metallothionein gene; the CD34 promoter; the CD8 promoter; the thymidine kinase (TK) promoter; the B19 parvovirus promoter; the PGK promoter; the glucocorticoid promoter; the heat shock protein (HSP) promoter; the immunoglobulin promoter; the MMTV promoter; the Rouss sarcoma virus (RSV) promoter; the lac promoter; and the CaMV promoter. The nucleic acids disclosed herein are selected from the 35S promoter; nopaline synthase promoter; MND promoter; and MNC promoter.

[0020] In embodiments, the Disclosure provides nucleic acids disclosed herein, wherein the promoter is a CMV promoter.

[0021] In embodiments, the Disclosure provides the nucleic acid disclosed herein, wherein the CMV promoter comprises nucleotides 150-812 of SEQ ID NO: 2.

[0022] In embodiments, the Disclosure provides nucleic acids disclosed herein in which an intronic sequence is operably linked to a sequence encoding an RdCVFL protein.

[0023] In embodiments, the Disclosure provides nucleic acids disclosed herein, wherein the intron sequence is a beta-globin intron sequence.

[0024] In embodiments, the Disclosure provides nucleic acids disclosed herein, wherein the intron sequence comprises nucleotides 820-1312 of SEQ ID NO: 2.

[0025] In embodiments, the Disclosure provides nucleic acids disclosed herein, wherein the nucleic acid comprises SEQ ID NO: 2, the nucleotide sequence 150-2044 of SEQ ID NO: 2, or the nucleotide sequences 150-812, 820-1312, and 1340-2044 of SEQ ID NO: 2.

[0026] In embodiments, the present disclosure provides vectors comprising nucleic acids disclosed herein.

[0027] In embodiments, the Disclosure provides vectors disclosed herein, wherein the vector is a nonviral vector.

[0028] In embodiments, the Disclosure provides vectors disclosed herein in which the nonviral vector is selected from lipid nanoparticles (LNPs), highly branched poly(β-aminoesters) (HPAEs), single-chain cyclic polymers (SCKPs), poly(amideamines) (PAMAM) dendrimers, and polyethyleneimines (PEIs).

[0029] In embodiments, the Disclosure provides vectors disclosed herein, wherein the vector is a viral vector.

[0030] In embodiments, the Disclosure provides vectors disclosed herein, wherein the viral vector is selected from retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, herpesvirus vectors, hepatitis virus vectors, SV40 vectors, EBV vectors, and Newcastle disease virus vectors.

[0031] In embodiments, the Disclosure provides vectors disclosed herein, wherein the viral vector is an adeno-associated virus (AAV) vector.

[0032] In an embodiment, the Disclosure provides a vector disclosed herein, wherein the AAV vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAVrh74.

[0033] In embodiments, the Disclosure provides a vector disclosed herein, wherein the AAV vector is AAV2.

[0034] In embodiments, the Disclosure provides a vector disclosed herein, wherein the AAV vector is AAV8.

[0035] In embodiments, the Disclosure provides vectors disclosed herein, wherein the viral vector is not a bovine immunodeficiency virus vector.

[0036] In embodiments, the present disclosure provides isolated cells comprising the nucleic acids disclosed herein, the cells being capable of secreting the RdCVFL protein.

[0037] In embodiments, the present disclosure provides a method for producing the RdCVFL protein, comprising culturing cells under conditions that enable the expression and secretion of the RdCVFL protein, and isolating the RdCVFL protein from the cell culture.

[0038] In embodiments, the present disclosure provides a method disclosed herein, further comprising purifying the RdCVFL protein from a cell culture.

[0039] In embodiments, the present disclosure provides a method for secreting RdCVFL protein from cells, comprising administering the nucleic acid or vector disclosed herein to cells under conditions that enable the expression and secretion of RdCVFL encoded by the nucleic acid or vector.

[0040] In embodiments, the Disclosure provides isolated cells or methods disclosed herein, wherein the cells are mammalian cells.

[0041] In embodiments, the present disclosure provides isolated cells or methods disclosed herein, wherein the cells are human cells.

[0042] In embodiments, the present disclosure provides isolated cells or methods disclosed herein, wherein the mammalian cells are ophthalmic cells.

[0043] In embodiments, the Disclosure provides isolated cells or methods disclosed herein, wherein the ophthalmic cells are selected from retinal pigment epithelial (RPE) cells, rod cells, cone cells, bipolar cells, horizontal cells, amacrine cells, ganglion cells, and ARPE-19 cells.

[0044] In embodiments, the present disclosure provides isolated cells or methods disclosed herein, wherein the cells are in vitro.

[0045] In embodiments, the present disclosure provides isolated cells or methods disclosed herein, wherein the cells are in vivo.

[0046] In embodiments, the Disclosure provides isolated cells or methods disclosed herein, wherein the cells are ex vivo.

[0047] In embodiments, the Disclosure provides isolated cells or methods disclosed herein, wherein the mammalian cells are selected from 293 cells, CHO cells, PerC6 cells, Vero cells, BHK cells, HeLa cells, COS cells, MDCK cells, 3T3 cells, and WI38.

[0048] In embodiments, the present disclosure provides isolated cells or methods disclosed herein in which cells are encapsulated.

[0049] In embodiments, the Disclosure provides pharmaceutical preparations comprising (i) a pharmaceutically acceptable carrier, (ii) a nucleic acid disclosed herein, a vector disclosed herein, or (iii) a combination thereof.

[0050] In embodiments, the present disclosure provides a method for preserving rod and cone cells in the eye of a mammal, comprising administering to the eye of a mammal an amount effective for preserving rod and cone cells the nucleic acids disclosed herein, the vectors disclosed herein, the pharmaceutical compositions disclosed herein, or a combination thereof.

[0051] In embodiments, the Disclosure provides a method disclosed herein in which a vector or nucleic acid is administered by subretinal injection.

[0052] In embodiments, this disclosure relates to approximately 5 × 10 AAV vectors. 8 ~Approx. 1×10 11 This invention provides a method for administering a vector genome copy (GC) by subretinal injection.

[0053] In embodiments, the Disclosure provides a method disclosed herein in which a vector or nucleic acid is administered by intravitreous injection, anterior chamber injection, subconjunctival injection, or sub-Tenon's capsule injection.

[0054] In embodiments, this disclosure relates to approximately 5 × 10 AAV vectors. 8 ~Approx. 5×10 12 This invention provides a method disclosed herein in which a vector genome copy (GC) is administered by intravitreal injection.

[0055] In embodiments, the present disclosure provides a method disclosed herein, wherein the mammal is a human.

[0056] In embodiments, the Disclosure provides methods disclosed herein for a mammal suffering from an ocular disease selected from the group consisting of retinal dystrophy, Stargardt disease, retinitis pigmentosa, age-related macular degeneration in dry state (dry AMD), geographic atrophy (an advanced stage of dry AMD), wet age-related macular degeneration (wet AMD), glaucoma / ocular hypertension, diabetic retinopathy, Valday-Biedl syndrome, Bassen-Kohnzweig syndrome, Best's disease, choroidopathy, cerebral gynecomastia, congenital amaurosis, Refsun syndrome, Usher syndrome, thyroid-related eye disease, Graves' disease, diseases related to retinal pigment epithelial cells, anterior segment disease, lens disease / cataract, eyecup syndrome, or uveitis.

[0057] In embodiments, the Disclosure provides a method disclosed herein in which, prior to administration, the stored rod and pyramidal cells do not contain the nucleic acids disclosed herein.

[0058] In embodiments, the Disclosure provides a method disclosed herein, comprising administering the nucleic acid or vector disclosed herein into the eye of a mammal, wherein the nucleic acid or vector is administered by subretinal injection, and rod and cone cells are preserved at a location at least 1 mm from the site of subretinal injection.

[0059] In an embodiment, the present disclosure provides a method for preserving rod cells at a location at least 2 mm from the site of subretinal injection.

[0060] In embodiments, the present disclosure provides a method for treating a disease, wherein the disease is a central nervous system (CNS) disease, comprising administering a nucleic acid, a vector, a pharmaceutical preparation, or a combination thereof to a mammal.

[0061] In embodiments, the Disclosure provides a method for treating the CNS disease disclosed herein, wherein the CNS disease is Alzheimer's disease, Huntington's disease, Parkinson's disease, or an olfactory disorder.

[0062] In embodiments, the Disclosure provides a method for treating the diseases disclosed herein, wherein the administration includes intra-arachnoid injection.

[0063] In embodiments, this disclosure relates to approximately 5 × 10 AAV vectors. 8 ~Approx. 5×10 14 This invention provides a method for treating a disease as disclosed herein, in which a vector genome copy (GC) is administered by intraarachnoid injection.

[0064] In embodiments, the present disclosure provides a method for treating the diseases disclosed herein, wherein the administration includes intravenous injection.

[0065] In embodiments, this disclosure relates to approximately 5 × 10 AAV vectors. 8 ~Approx. 1×10 15 This invention provides a method for treating a disease as disclosed herein, in which a vector genome copy (GC) is administered by intravenous injection.

[0066] In embodiments, the present disclosure provides a method for treating a disease disclosed herein, wherein the mammal is a human.

[0067] This summary of the present invention does not necessarily describe all or necessary features of the present invention. The present invention may also exist in subcombinations of the described features. [Brief explanation of the drawing]

[0068] For illustrative purposes, the drawings show specific embodiments of the present invention. However, the present invention is not limited to the exact arrangement and means of the embodiments shown in the drawings.

[0069] [Figure 1] This shows the SDS-PAGE silver staining analysis of purified recombinant AAV2-hIGK-hRdCVF1L vector particles. The presence of AAV particles was confirmed by visualization of VP1, VP2, and VP3 capsid proteins. [Figure 2]This shows Western blot analysis of RdCVF1L expression and secretion in ARPE-19 cells transduced with the rAAV-RdCVF1L vector. A protein double chain immunoreactive to anti-RdCVF1L antibody was detected at approximately 30 kDa (close to the theoretical molecular weight of hRdCVF1L) in cell lysates and culture medium from ARPE-19 cell cultures transduced with rAAV2.hIgK.hRdCVF1L, but was not detected with the control vector rAAV2.EGFP. The higher molecular weight band of the RdCVF1L double chain represents the glycosylated form of RdCVF1L. [Figure 3] This report presents Western blot analysis of RdCVF1L expression and secretion in HEK293 cells transduced with the rAAV-RdCVF1L vector, mediated by two different signal sequences. A protein double chain immunoreactive to anti-RdCVF1L antibodies was detected at approximately 30 kDa (close to the theoretical molecular weight of hRdCVF1L) in cell lysates and culture media from HEK293 cultures transduced with rAAV2.mIgK.hRdCVF1L, rAAV2.hIgK(-D).hRdCVF1L, and rAAV2.hIgK.hRdCVF1L, but was not detected with the control vector rAAV2.EGFP. The expression and secretion of RdCVF1L mediated by the rAAV2.mIgK.hRdCVF1L and rAAV2.hIgK.hRdCVF1L vectors were far more efficient than that of the rAAV2.hIgK(-D).hRdCVF1L vector. rAAV2.mIgK.hRdCVF1L is an AAV vector encoding human RdCVF1L along with a mouse IgK signaling sequence; rAAV2.hIgK(-D).hRdCVF1L is an AAV vector encoding human RdCVF1L along with a human IgK signaling sequence (SEQ ID NO: 8); and rAAV2.hIgK.hRdCVF1L is an AAV vector encoding human RdCVF1L along with a human IgK signaling sequence (+Asp) (SEQ ID NO: 7). [Figure 4]This study demonstrates photoreceptor rescue by subretinal injection of AAV-RdCVF1L in rd10 mice. Mouse retinal sections showed rescue of photoreceptor cells in the AAV2-RdCVF1L-treated eye (left panel) compared to the untreated contralateral eye (right panel). Arrows indicate the location of photoreceptor cells (outer nuclear layer, ONL). The treated eye had five ONL layers (left panel), while the untreated contralateral eye had only one ONL layer (right panel). [Figure 5] This study demonstrates that RdCVF1L reduced Aβ1-42-induced toxicity in SH-SY5Y cells. LDH release was measured in culture medium and cell lysates. The LDH ratio (culture medium / total (culture medium + lysates)) was normalized to the percentage of the vehicle control. SH-SY5Y cells were incubated for 24 hours with vehicle RdCVF1L (175, 350 ng / mL) in serum-free medium without 0, 2.5, 5, and 10 μM β-amyloid 1-42. LDH was measured in culture medium and cell lysates. LDH ratio (culture medium / total) normalized to the percentage of the vehicle, two-way ANOVA, +++ P<0.001, vs. vehicle, no β-A; *** P<0.001, vs. vehicle containing 10 μM β-A. [Figure 6] This study demonstrates that RdCVF1L reduced Aβ1-42-induced toxicity in NHNP cells. LDH release was measured in culture medium and cell lysates. The LDH ratio (culture medium / total (culture medium + lysates)) was normalized to the percentage of the vehicle control. Differentiated NHNP cells (8 days) were incubated with the vehicle RdCVF1L (100, 200 ng / mL) for 2 days in 0 and 10 μM culture medium + 1% FBS (β-A1-42-free) (Invitrogen, cat.03-112). LDH was measured in culture medium and cell lysates. LDH ratio (culture medium / total) normalized to 1% of the vehicle, two-way ANOVA, +++<0.001, vs. vehicle, no β-A; ***P<0.001, vs. vehicle containing 10 μM β-A. [Modes for carrying out the invention]

[0070] As used herein, the transitional term “including” is non-limiting. Claims using this term may include elements in addition to those enumerated in such claims. Thus, for example, a claim can be read as a method that includes other steps not specifically enumerated herein, insofar as the enumerated elements or their equivalents exist.

[0071] The terms "identity" and "identical" refer to the percentage of matching sequences between two sequences when used in the context of comparing two sequences, such as nucleotide or amino acid sequences. Percent identity can be determined by algorithms commonly used by those skilled in the art. For example, percentage identity can be determined using tools and programs available from the National Center for Biotechnology Information (NCBI), as available on the NCBI website. The percentage identity of two nucleotide sequences can be determined, for example, using the NCBI / BLAST / blastn suite. blastn can be used with the following parameters: expect threshold=10, word size=28, max matches in a query range=0, match / mismatch scores=1,-2, gap costs=existence:5 extension:2.

[0072] Any method or composition described herein is considered applicable in relation to any other method or composition described herein. When used in conjunction with the claims and / or the term “including” herein, the use of the words “a” or “an” may mean “one,” but also coincides with the meanings of “one or more,” “at least one,” and “one or more.” When used with a list, the use of the term / phrase “and / or” means that one or more of the listed items may be available, and is not limited to, for example, one or all of the elements.

[0073] Introduction Individuals with some form of retinal dystrophy have been found to have lower levels of intraocular RdCVF protein than individuals without retinal dystrophy (see, e.g., PCT Publication WO02 / 081513). Different forms of RdCVF protein promote the survival of cone photoreceptor cells in vitro and in vivo. For example, intraocular injection of a short form of human RdCVF1 (RdCVF1S) protein not only rescued cone cells from degeneration but also preserved their function in an animal model of hereditary retinal degeneration (Yang et al. (2009) Mol Therapy 17:787-795).

[0074] Despite the potential of the RdCVF protein for the treatment of various diseases, including eye diseases, expressing significant levels of RdCVF from gene therapy vectors on a large scale has been challenging (see, for example, U.S. Patent Publication 2011 / 0034546, paragraph

[0004] ). In fact, the protein's hydrophobicity has hindered measurable production and purification using standard methods. See, for example, Sahel JA, Novel Treatments for Vision Disorders. Research EU Results Magazine. September 2014 (cordis.europa.eu / project / id / 241683 / reporting).

[0075] This disclosure provides nucleic acids and vectors encoding the RdCVF protein along with a signal sequence, as well as compositions and therapeutic methods utilizing them, enabling the large-scale production of RdCVF for therapeutic use. In embodiments of this disclosure, the large-scale production of various forms of RdCVF is achieved by nucleic acids encoding the RdCVF protein and a remarkably excellent human immunoglobulin kappa chain (IgK) signal sequence. In embodiments, the use of a human IgK sequence results in the expression of a polypeptide with remarkably excellent expression and secretion, which was observed as a double helix representing glycosylated and non-glycosylated forms of RdCVF1L in Western blot analysis. This is important because glycosylation is a post-translational modification that indicates the protein is secreted through the endoplasmic reticulum and Golgi secretory pathways. In embodiments, the human IgK signal sequence comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7 or SEQ ID NO: 8. In embodiments, the human IgK signal sequence comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 7. In embodiments, the disclosure provides nucleic acids encoding a human RdCVF protein and a human IgK signal sequence that is not a naturally occurring protein.

[0076] In one embodiment, the nucleic acid of the present invention encodes an RdCVF1 protein or an RDCVF2 protein and an IgK signal sequence containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 7.

[0077] In an embodiment, the disclosure provides a nucleic acid encoding a protein comprising human RdCVF1L protein (amino acids 24-234 of SEQ ID NO: 3) and a human IgK signal sequence, wherein the human IgK signal sequence comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 7.

[0078] In an embodiment, the present disclosure provides a nucleic acid encoding a protein comprising the RdCVF2L protein (SEQ ID NO: 13) and an IgK signal sequence having at least 95% sequence identity with SEQ ID NO: 7.

[0079] In one embodiment, the nucleic acid of the present invention encodes a long RdCVF1 protein and an IgK signal sequence containing the amino acid sequence of SEQ ID NO: 7. In another embodiment, the nucleic acid of the present invention encodes a long RdCVF2 protein and an IgK signal sequence containing the amino acid sequence of SEQ ID NO: 7.

[0080] In embodiments, the disclosure provides nucleic acids encoding an RdCVF protein and a human IgK signaling sequence comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 3.

[0081] In embodiments, the disclosure provides a nucleic acid encoding an RdCVF protein and a human IgK signal sequence comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 15.

[0082] In an embodiment, the present disclosure provides a nucleic acid encoding a protein comprising the RdCVF1L protein (amino acids 24-234 of SEQ ID NO: 3) and an IgK signal sequence having at least 95% sequence identity with SEQ ID NO: 9.

[0083] PCT Publications WO2002 / 081513, WO2008 / 148860, WO2009 / 146183, and WO2013 / 63383 describe various compositions and methods related to RdCVF. In some cases, the RdCVF-related compositions and methods described in PCT Publications WO2002 / 081513, WO2008 / 148860, WO2009 / 146183, and WO2013 / 63383 can be utilized, for example, by replacing the nucleic acid, vector, or protein encoding RdCVF with that of the present invention (e.g., nucleic acid and vector containing an RdCVF coding sequence and a human IgK signaling sequence).

[0084] RdCVF The RdCVF protein promotes the survival of cone photoreceptor cells in vitro and in vivo. For example, intraocular injection of a short form of human RdCVF1 (RdCVF1S) protein not only rescued cone cells from degeneration but also preserved their function in an animal model of hereditary retinal degeneration (Yang et al. (Mol Therapy (2009) 17:787-795 and supplemental material)). RdCVF is expressed by several cell types, including rod photoreceptor cells in the retina (Leveillard et al. (2004) Nature Genetics 36:755-759).

[0085] Two distinct RdCVF genes are found in humans and other mammals, and they are called RdCVF1 and RdCVF2. Both RdCVF genes encode two products via alternative splicing (a full-length protein and a C-terminus shortened protein, respectively, known as RdCVF-long ("RdCVFL")) and RdCVF-short. As used herein, "RdCVF1L" refers to the full-length RdCVF1 protein (amino acids 24-234 of SEQ ID NO: 3), and "RdCVF2L" refers to the full-length RdCVF2 protein (SEQ ID NO: 13).

[0086] In some embodiments, this disclosure provides recoded RdCVF coding sequences. Recoded RdCVF coding sequences can encode any RdCVF protein, including any of those disclosed herein. Sequences of various RdCVF proteins are described in PCT Publications WO2002 / 081513 and WO2010 / 029130, Chalmel et al. (BMC Molecular Biology (2007) 8:74 pp1-12 and supplemental information), Leveillard et al. (Nature Genetics (2004) 36:755-759 and supplemental information), Yang et al. (Mol This can be found in Therapy (2009) 17:787-795 and supplementary materials, as well as in GenBank registration numbers NP_612463, AAH14127, Q96CM4, EAW84608, CAD67528, Q5VZ03, NP_001155097, NP_660326, CAM24748, CAM14247, AAH22521, and CAD67531.

[0087] In some embodiments, RdCVF proteins are fragments or analogs of RdCVF proteins that maintain the viability or protective effect of pyramidal and / or rod cells. Methods for measuring these activities or effects are well known in the art. For example, Leveillard et al. (Nature Genetics (2004) 36:755-759 and supplemental information) describe relevant mouse models and in vitro methods for detecting RdCVF activity. Nucleic acid-encoded RdCVF proteins or RdCVF may have amino acid sequences other than those found in nature. For example, non-natural RdCVF proteins may contain amino acids (e.g., at the amino-terminus or carboxy-terminus) in addition to those found in naturally occurring RdCVF proteins, and / or may contain one or more amino acid substitutions (e.g., conserved or non-conserved amino acid substitutions) compared to the naturally occurring RdCVF amino acid sequence. Conservative amino acid substitutions should generally not substantially alter the structural properties of the parent sequence (for example, the substituted amino acid should not tend to disrupt helices in the parent sequence or other types of secondary structures that characterize the parent sequence). Examples of polypeptide secondary and tertiary structures recognized in the art are described in Proteins, Structures and Molecular Principles (Creighton, Ed., WH Freeman and Company, New York (1984)), Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)), and Thornton et al. Nature 354:105 (1991).Conservative substitutions include, but are not limited to, those from the following groups: acidic residues Asp(D) and Glu(E); basic residues Lys(K), Arg(R), and His(H); hydrophilic uncharged residues Ser(S), Thr(T), Asn(N), and Gln(Q); aliphatic uncharged residues Gly(G), Ala(A), Val(V), Leu(L), and Ile(I); nonpolar uncharged residues Cys(C), Met(M), and Pro(P); aromatic residues Phe(F), Tyr(Y), and Trp(W); alcohol-containing residues S and T; aliphatic residues I, L, V, and M; cycloalkenyl groups Synthetic residues F, H, W, and Y; hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Y; negatively charged residues D and E; polar residues C, D, E, H, K, N, Q, R, S, and T; positively charged residues H, K, and R; small residues A, C, D, G, N, P, S, T, and V; very small residues A, G, and S; residues involved in turn formation, A, C, D, E, G, H, K, N, Q, R, S, P, and T; and flexible residues Q, T, K, S, G, P, D, E, and R. In some embodiments of the present invention, the unnaturally occurring RdCVF protein has additional amino acids at the amino terminus, e.g., additional amino acids from a signal peptide. In some embodiments, the RdCVF protein of the present invention is first translated from a nucleotide coding sequence having a signal peptide, and in some cases, all or some of the amino acids of the signal peptide are retained on the expressed and / or secreted RdCVF protein of the present invention.

[0088] Re-coded RdCVF code sequence In embodiments, the disclosure provides a nucleic acid comprising a nucleotide sequence encoding an RdCVF protein and a human IgK signal sequence, wherein the human IgK signal sequence comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 7 or SEQ ID NO: 8, and the RdCVF coding sequence comprises a re-encoded nucleotide sequence. In embodiments, the RdCVF re-encoded nucleotide sequence lacks an start methionine codon.

[0089] The terms “recoded” or “recoded nucleotide sequence” mean that at least one native codon has been replaced with another codon that codes for the same amino acid as the native codon. In some embodiments, a recoded RdCVF coding region has at least 2.5%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least or at least 95% of recoded codons. In some embodiments, about 20–50%, 35–45%, 38–42%, or 39–41% or codons are recoded. In some embodiments, the recoded codons are replaced with codons that are more commonly used in humans. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55% of codons are replaced with codons that are more commonly used in humans.

[0090] In some embodiments, the recoded sequence has approximately 70–90%, 75–85%, 80–85%, or 82–85% identity with the corresponding native coding sequence. In some embodiments, the recoded nucleotide sequence differs by at least 15% of nucleotides compared to the corresponding native nucleotide sequence. In some embodiments, the recoded nucleotide sequence is less than 90% identical to the corresponding native nucleotide sequence.

[0091] Furthermore, recoding can also be used to alter the chemical composition of DNA and / or RNA coding sequences, such as the guanine / cytosine (GC) ratio. In some embodiments, recoding of the RdCVF coding region increases the GC content to at least 60%. In some embodiments, the recoded RdCVF coding region has a GC ratio of 60–64% or 60.4%–63.5%.

[0092] Recoding can be used to alter the secondary structure of mRNA. Recoding can also be used to remove or add specific motifs or sites, such as procariatic inhibitory motifs, consensus splice donor sites, latent splice donor sites, or combinations thereof, to the coding sequence or nucleic acid molecule. In some embodiments, the recoded RdCVF coding sequence has fewer procariatic inhibitory motifs, consensus splice donor sites, latent splice donor sites, or combinations thereof than the native sequence. In some embodiments, the recoded RdCVF coding sequence does not contain procariatic inhibitory motifs, consensus splice donor sites, and / or latent splice donor sites.

[0093] Hoover et al. (Nucleic Acids Res. (2002) 30:e43, pp1-7), Fath et al. (PLoS one (2011) 6:e17596 pp1-14), Graf et al. (J Virol (2000) 74:10822-10826), Raab et al. (Syst Synth Biol (2010) 4:215-225), and U.S. Patent Application No. 2007 / 0141557 describe the recoding of the coded area.

[0094] In some embodiments of the present invention, the recoded RdCVF coding sequence does not contain the original RdCVF ATG codon and / or RdCVF stop codon (e.g., TAG). For example, the recoded RdCVF coding sequence can be operably ligated at the 5' or 3' end to another coding sequence, resulting in a protein containing heterologous amino acid sequences as the N-terminus and / or C-terminus, respectively, relative to the RdCVF amino acid sequence. In some of these embodiments, the original RdCVF ATG codon and / or RdCVF stop codon may be deleted or present in the RdCVF coding region. See, for example, SEQ ID NOs: 3 and SEQ ID NOs: 6. If another coding sequence is fused in-frame at the 3' end of the RdCVF coding region, the native RdCVF stop codon is typically not present at the end of the RdCVF coding sequence.

[0095] As used herein, the term “operatably linked” (or “operatably linked”) means, in relation to the parallel arrangement of two or more components (such as sequence elements), that the components are arranged in such a way that both components may function properly and that at least one of the components may mediate a function exerted on at least one of the other components. For example, a nucleic acid operatably linked to a promoter is under the functional control of that combination, e.g., the transcription initiation regulation of the promoter.

[0096] In some embodiments, the recoded RdCVF coding sequence is the recoded sequence that codes for amino acids 24-234 of SEQ ID NO: 3.

[0097] Signal peptides / secretion signals The protein secretion pathway is utilized by more than a quarter of the human proteome. Signal peptides play a crucial role in inducing newly synthesized proteins to move from the cytosol to another location via the secretory pathway (e.g., the cell membrane for transmembrane proteins or the extracellular space for secretory proteins). Signal peptides are extremely diverse. In humans alone, more than 3,000 proteins containing various signal peptides have been identified to date. The highly diverse primary sequences of these signal peptides suggest that they play a role in regulating the physiological levels of secretion of specific proteins from cells (Kober, L. (2013) Biotechnol. Bioeng., 110:1164-1173, Cho, HJ. (2019) J. Microbiol. Biotechnol., 29:304-310, Liaci and Forster (2021) Int. J. Mol. Sci. 2021, 22, 11871. http: / / doi.org / 10.3390 / ijms222111871, Kangro, K. (2022) J. Thromb. Haemost., 20:2379-2385). The expression and secretion levels of a given protein depend heavily on the selection of the signal peptide (Knappskog, S. (2007) Biotechnology, 128:705-715). Simply placing a heterologous signal peptide adjacent to a mature protein sequence does not guarantee secretory expression, and protein expression may be completely suppressed when placed directly adjacent to a non-native signal peptide (Guler-Cane, G (2016) PLOS ONE | DOI:10.1371 / journal.pone.0155340 May 19, 2016). To make matters even more complex, the efficiency of the signal peptide in inducing protein secretion is also influenced by the amino acids of the mature protein.

[0098] Endogenous human RdCVF lacks a identifiable signal peptide. A mouse IgK signal peptide has been demonstrated to mediate the efficient secretion of RdCVFL (US9,265,813). Using a mouse IgK signal sequence, the potential immunogenicity of the human signal sequence was reduced in in vivo studies in mice. Since there is no significant homology between mouse and human IgK signal peptides, this disclosure provides a human IgK signal peptide capable of mediating the expression and secretion of RdCVFL. The use of human-derived signal peptides is expected to avoid the immunogenicity associated with mouse IgK signal peptides when gene therapy vectors are tested in human studies.

[0099] The signal sequence is translated in-frame as a peptide typically bonded to the amino terminus of the optimal polypeptide. The secretory signal sequence interacts with host cell mechanisms, triggering the secretion of polypeptides from the cell. As part of the secretory process, this secretory signal sequence is typically cleaved or at least partially cleaved. The term “signal sequence” also refers to the nucleic acid sequence encoding the signal peptide.

[0100] The structure of a typical signal peptide can include the following three distinct regions: (i) an N-terminal region containing a number of positively charged amino acids (e.g., lysine and arginine), (ii) a central hydrophobic core region (h-region), and (iii) a hydrophilic cleavage region (c-region) containing a sequence motif recognized by a signal peptidase. (See, for example, von Heijne, G. (1983) Eur. J. Biochem., 133:17-21, von Heijne, G. (1985) J. Mol. Biol., 184:99-105, von Heijne, G. (1997) Protein Engineering (10): 1-6). These signal peptides can be used according to the present invention. In some embodiments, the signal peptide is derived from an immunoglobulin such as IgK.

[0101] The signal sequence can be a mammalian, mouse, or human signal sequence. In some embodiments, the nucleic acid or vector of the present invention contains nucleotides 1340-1408 of SEQ ID NO: 2 or 1340-1405 of SEQ ID NO: 2. In some embodiments, the signal sequence encodes an amino acid sequence containing amino acids 1-23 of SEQ ID NO: 3, or contains amino acids 1-22 of SEQ ID NO: 4. The nucleotide sequence encoding the signal peptide can be a wild-type sequence or a re-encoded sequence.

[0102] In some embodiments of the present invention, the signal peptide sequence is operably ligated to the N-terminus or C-terminus of RdCVF (e.g., RdCVF1L or RdCVF2L). In some embodiments, the signal peptide directs the translocation of a protein into the secretory pathway (e.g., into the endoplasmic reticulum (ER)). In some embodiments, the signal peptide facilitates protein transport from the cytoplasm to an extracellular destination. The signal peptide sequence may be selected from naturally occurring signal peptide sequences, derivatives thereof, or synthetically designed sequences. In some embodiments, non-limiting parameters of the designed signal peptide sequence include a 3-40 residue sequence comprising a 3-20 residue hydrophobic core with several relatively hydrophilic residues positioned laterally.

[0103] nucleic acid The present invention includes nucleic acids comprising nucleotide sequences encoding RdCVF, and vectors comprising these nucleic acids.

[0104] To ensure local and / or long-term expression of the target nucleic acid, some embodiments of the present invention intend to transduce cells with a nucleic acid or vector encoding RdCVF. The present invention should not be construed as being limited to any one specific nuclear delivery method, and the use of any available nucleic acid delivery vehicle or engineered cells with either an in vivo or in vitro nucleic acid delivery strategy (see, for example, U.S. Patents 6,231,879, 6,262,034, 6,264,941, 6,303,136, 6,322,804, 6,436,427, and 6,878,544, such as Neurotech, Lincoln, and RI technologies) and the nucleic acid of the present invention encoding RdCVF itself (e.g., "naked DNA") may be used in carrying out the present invention. Various delivery vehicles, such as vectors, may be used in conjunction with the present invention. For example, viral vectors, amphiphilic lipids, cationic polymers such as polyethyleneimine (PEI) and polylysine, dendrimers such as comb-burst molecules and star-burst molecules, nonionic lipids, anionic lipids, vesicles, liposomes, and other synthetic nucleic acid delivery methods (see, for example, U.S. Patents No. 6,958,325 and 7,098,030, Langer, Science 249:1527-1533 (1990), "Liposomes" in "The Therapy of Infectious Disease and Cancer" by Treat et al., and Lopez-Berestein & Fidler (eds.), Liss, New York, pp. 317-327 and 353-365 (1989), Wang et al. J Nanobiotechnol 21,272 (2023)), "naked" nucleic acids, etc., can be used in carrying out the present invention.

[0105] In some embodiments, nucleic acid molecules are used in which the RdCVF coding sequence and any other desired sequence are located lateral to a region that promotes homologous recombination at a desired site in the genome, thus resulting in intrachromosomal expression of the RdCVF nucleic acid (Koller et al., (1989) Proc.Natl.Acad.Sci.USA86:8932-8935, Zijlstra et al. (1989) Nature 342:435-438). Delivery of the nucleic acid to the patient may be direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector, or it may be indirect, in which case cells are first transformed with the nucleic acid in vitro and then transplanted into the patient.

[0106] A vector is a means for introducing a target nucleic acid (e.g., a therapeutic nucleic acid capable of encoding a therapeutic protein) into target cells. Methods for obtaining or constructing a target vector include, but are not limited to, standard gene manipulation techniques, sequencing reactions, restriction enzyme digestion, polymerase reactions, PCR, PCR SOEing, ligation, recombinase reactions (e.g., Invitrogen's GATEWAY® technology), other enzymes active to nucleic acids, materials and methods for bacterial and viral growth, chemicals and reagents, site-directed mutagenesis protocols, etc. See, for example, Maniatis et al., text, "Molecular Cloning". The nucleic acids of the present invention typically include a promoter sequence operably ligated to a human IgK signaling sequence and a human RdCVF coding sequence. The promoter may be, for example, a tissue-specific promoter, a cell-specific promoter, an inducible promoter, an inhibitory promoter, a constitutive promoter, a synthetic promoter, or a hybrid promoter. Examples of promoters useful in the components of the present invention include, but are not limited to, the following: phage-lambda (PL) promoter; SV40 early promoter; herpes simplex virus (HSV) promoter; cytomegalovirus (CMV) promoters such as the human CMV pre-early promoter; hybrid promoters including the CMV enhancer and chicken beta-actin promoter; tetracycline-regulated transactivator-responsive promoter (tet) systems; long-terminal repeat (LTR) promoters such as MoMLV LTR, BIV LTR, or HIV LTR; Moloney's mouse sarcoma virus U3 region promoter; granzyme A promoter; regulatory sequences of the metallothionein gene; CD34 promoter; CD8 promoter; thymidine kinase (TK) promoter; B19 parvovirus promoter; PGK promoter; glucocorticoid promoter; heat shock protein (HSP) promoters such as HSP65 and HSP70 promoters; immunoglobulin promoter; MMTV promoter; Rouss sarcoma virus (RSV) promoter; lac promoter; CaMV 35S promoter; and nopaline synthase promoter. In some embodiments, the promoter is the MND promoter (Robbins et al., 1997, J. Virol. 71:9466-9474) or the MNC promoter, the MNC promoter being a derivative of the MND promoter in which the LTR enhancer is combined with a CMV promoter with minimal enhancement (Haberman et al., J. Virol. 74(18):8732-8739, 2000). In some embodiments, the RdCVF coding sequence is operably ligated to a promoter sequence containing nucleotide sequences 150-812 of SEQ ID NO: 2.

[0107] In some embodiments, the vector or nucleic acid of the present invention includes an intron operatively ligated to the coding sequence of the RdCVF protein. The intron may be derived from the RdCVF gene or may be a heterologous intron. Heterologous introns are well known, and non-limiting examples include the human β-globin gene intron and the β-actin intron. In some embodiments, the intron sequence is the human β-globin gene intron sequence. In some embodiments, the intron sequence includes nucleotides 820-1312 of SEQ ID NO: 2.

[0108] In some embodiments, the nucleic acid of the present invention comprises a nucleotide sequence encoding the coding sequence of the RdCVF protein, and the RdCVF coding sequence comprises a re-encoded nucleotide sequence. The nucleic acid can encode the RdCVF1 protein and / or the RdCVF2 protein. In some embodiments, the RdCVF protein is the RdCVF1-long (RdCVF1L) or RdCVF2-long (RdCVF2L) protein. In some embodiments, the RdCVF protein is the human RdCVF1-long or RdCVF2-long protein.

[0109] Typically, mammalian nucleotide coding regions begin with the nucleotide sequence ATG (start methionine codon), as seen in the human RdCVF coding region. As discussed herein, some embodiments of the invention provide a recoded RdCVF coding region, and in some further embodiments, the coding region is fused in-frame with a second coding region (e.g., the coding sequence of a signal sequence). In some of these cases, the ATG nucleotide sequence is not necessarily at the beginning of the RdCVF coding region; for example, the RdCVF coding region begins by encoding the second amino acid of a particular RdCVF protein. However, the ATG nucleotide sequence can be at the beginning of the RdCVF coding region even when the RdCVF coding region is operably ligated to another coding region at 5' relative to the RdCVF coding region.

[0110] In some embodiments, the nucleic acid of the present invention comprises SEQ ID NO: 2. In some embodiments, the nucleic acid of the present invention comprises nucleotides 150-812, 820-1312, and 1340-2044 of SEQ ID NO: 2.

[0111] In some embodiments, the nucleic acid of the present invention includes a coding region of RdCVF, and the RdCVF coding sequence is re-coded.

[0112] In some embodiments of the present invention, the nucleic acid of the present invention is contained within a vector such as a viral vector.

[0113] Viral vector The present invention includes a viral vector comprising the nucleic acid of the present invention (for example, a vector comprising a nucleic acid comprising a nucleotide sequence encoding the RdCVF protein together with a human IgK signal sequence), wherein the human IgK signal sequence comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 7 or SEQ ID NO: 8. Examples of viral vectors useful in the present invention are described in PCT Publication WO08 / 106644 and U.S. Patent Publication 2010 / 0120665. In some embodiments, the present invention is not limited to a specific viral vector. Viral vectors include, but are not limited to, retroviral vectors, lentiviral vectors, adenovirus vectors (see, e.g., U.S. Patent No. 7,045,344), AAV vectors (see, e.g., U.S. Patent No. 7,105,345), herpesvirus vectors (see, e.g., U.S. Patents No. 5,830,727 and 6,040,172), hepatitis (e.g., hepatitis D) virus vectors (see, e.g., U.S. Patent No. 5,225,347), SV40 vectors, EBV vectors (see, e.g., U.S. Patent No. 6,521,449), and Newcastle disease virus vectors (see, e.g., U.S. Patents No. 6,146,642, 7,442,379, 7,332,169, and 6,719,979). In some embodiments, the lentiviral vector is an HIV, EIAV, SIV, FIV, or BIV vector. In some embodiments, the vector is selected from AAV vectors or adenovirus vectors.

[0114] Furthermore, the present invention provides cells that produce the viral vector of the present invention. In embodiments, the cells that produce the viral vector of the present invention are 293 cells, CHO cells, PerC6 cells, Vero cells, BHK cells, HeLa cells, COS cells, MDCK cells, 3T3 cells, and WI38 cells.

[0115] The vector virion of the present invention may be administered to cells (e.g., mammalian cells) in vivo or in vitro. The vector (viral or nonviral) can be used to transduce or transform cells, including but not limited to undifferentiated cells, differentiated cells, somatic cells, primitive cells, and / or stem cells.

[0116] In some embodiments, the viral vector of the present invention includes a degradation promoter (DAF). For example, an enveloped viral vector includes a DAF on the viral membrane. In some embodiments, the DAF is a wild-type DAF. In some embodiments, the DAF is part of a fusion protein with the envelope protein (see, for example, Guibinga et al. Mol Ther. 2005 11(4):645-51).

[0117] Adenoviruses are non-enveloped nuclear DNA viruses with a genome typically of about 36 kb. Human adenoviruses are divided into numerous serotypes (approximately 47 types, numbered accordingly and classified into six groups: A, B, C, D, E, and F).

[0118] Recombinant adenovirus vectors possess tropism to both dividing and non-dividing cells, minimal pathogenicity, the ability to replicate to high titers for vector stock preparation, and the potential to carry relatively large nucleotide sequence inserts (Berkner, (1992) Curr. Top. Micro. Immunol. 158:39-66, Jolly, (1994) Cancer Gene Therapy 1:51-64). Adenovirus vectors with deletions of various adenovirus gene sequences have been designed as vehicles suitable for nucleic acid delivery to cells. In some embodiments, the adenovirus vectors of the present invention are helper-dependent or "gutless" adenovirus vectors. Adenovirus vectors with deletions of one or more of the following genes can be used: E1a, E1b, E2a, E2b, and E3. Methods for delivering adenovirus-based nucleic acids are described, for example, in U.S. Patents No. 5,824,544, No. 5,868,040, No. 5,871,722, No. 5,880,102, No. 5,882,877, No. 5,885,808, No. 5,932,210, No. 5,981,225, No. 5,994,106, No. 5,994,132, No. 5,994,134, and No. 6,001,557.

[0119] AAV vectors are obtained from single-stranded (ss)DNA parvovirus. A single AAV particle can accommodate up to 5 kb of ssDNA, leaving approximately 4.5 kb for the transgene and regulatory elements. For example, the trans-splicing system described in U.S. Patent No. 6,544,785 can nearly double this limit, and these types of vectors can also be used in conjunction with the present invention. In essence, any serotype of AAV can be used with respect to the present invention. In some embodiments of the present invention, serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrh74 may be used (for example, U.S. Patents 5,173,414, 5,252,479, 5,552,311, 5,658,776, 5,658,785, 5,763,416, 5,773,289, 5,843,742, 5,869,040, 5,942,496, 5,948,675, 6,001,650, and 7,790,449, PCT Publication WO2009 / 134681, Kassim et al.). See al., PLoS ONE (2010) 5(10)e13424:1-10, Kotin, Hum Mol Genet (2011) 20(R1):R2-6, and Shoti et al., Mol Ther Methods Clin Dev (2023) 31:101147. The present invention is not limited to these serotypes (see, for example, Gao et al. (2002) PNAS 99:11854-11859, and Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003).

[0120] Furthermore, the AAV vector of the present invention may be pseudotyped. The pseudotyped AAV vector contains the genome of one AAV serotype within the capsid of a second AAV serotype (see, for example, Auricchio et al., (2001) Hum. Mol. Genet., 10(26):3075-81). The AAV vector of the present invention may contain a mutated capsid and / or be retargeted. See, for example, Grieger et al. (Adv Biochem Eng Biotechnol (2005) 99:119-45), Goncalves et al. (Mol Ther. (2006) 13(5):976-86), and Warrington et al. (J Virol. (2004) 78(12):6595-609).

[0121] In some embodiments of the present invention, AAV vectors are coated with a polymer (e.g., a reactive polymer) to reduce the innate directivity or binding of the AAV vector, to retarget the AAV vector, and / or to provide resistance to neutralizing antiserum. See, for example, Carlisle et al. (J Gene Med. (2008) 10(4):400-11).

[0122] Furthermore, to enhance the transduction efficiency of the vector, alter its targeting of tissues and organs, reduce immunogenicity, and evade neutralizing antibodies against the viral vector, AAV vectors derived from various serotypes of AAV can be modified in the capsid protein by incorporating peptides. AAV capsid variable regions IV and VIII are tolerant of capsid modification (Havlik et al., J. Virology, 94:e00976 (2020), Havlik et al., J. Virology, 95:e0058721 (2021), Becker et al., Pathogens, 11:756 (2022), Gonzalez et al., Nat Commun., 13:5947 (2022)).

[0123] Retroviruses are RNA viruses whose viral genome is RNA. When a host cell is infected with a retrovirus, the genomic RNA is reverse transcribed into a DNA intermediate that is efficiently integrated into the chromosomal DNA of the infected cell. Lentiviruses contain other genes that have regulatory or structural functions. The use of retroviral vectors for gene delivery is described, for example, in U.S. Patent Nos. 6,013,516 and 5,994,136. Examples of BIV systems are described, for example, in Matukonis et al., 2002 Hum.Gene Ther. 13, 1293-1303, Molina et al., 2002 Virology. 304, 10-23, Molina et al., 2004 Hum.Gene Ther., 15, 65-877, U.S. Patent Nos. 6,864,085, 7,125,712, and 7,153,512, PCT Publication WO08 / 106644, and U.S. Patent Application No. 2010 / 0120665.

[0124] DNA viral vectors are viral vectors based on or derived from viruses that have a DNA-based genome. Non-enveloped viral vectors are viral vectors based on or derived from viruses that lack a lipid bilayer membrane.

[0125] In some embodiments, the viral vector of the present invention is an AAV vector. In some embodiments, the viral vector of the present invention is not a bovine immunodeficiency virus vector or a lentiviral vector. In some embodiments, the viral vector is selected from the group consisting of DNA viral vectors, non-enveloped viral vectors, and adenovirus vectors.

[0126] The remarkable success of lipid nanoparticle (LNP) delivery technology in COVID-19 vaccines has demonstrated the usefulness of LNPs as a non-viral vector delivery platform. Furthermore, LNPs have been used in various preclinical studies to deliver mRNA, siRNA, antisense oligonucleotides, microRNA, and DNA (Hald Albertsen et al., Adv Drug Deliv Rev. 188:114416 (2022)). Nucleic acid sequences encoding RdCVF may be deliverable using LNP technology.

[0127] Cell delivery of RdCVF, including inclusion cells. Another approach to gene therapy or protein delivery involves introducing genes into cells in vitro or ex vivo, followed by administering the cells to a mammal or patient. The introduction of nucleic acids into cells can be carried out by any method, including transfection, microinjection, electroporation, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, lipofection, particulate gun, calcium phosphate-mediated transfection, viral vector, or bacteriophage transduction. Optionally, selectable markers can also be introduced into the cells. If selectable markers are available, cells can be selectively isolated / selected, for example, to enhance expression and / or to isolate / select cells expressing introduced coding regions (see, e.g., Loeffler & Behr, Meth. Enzymol. 217:599-618 (1993), Cohen et al., Meth. Enzymol. 217:618-644 (1993), and Cline, Pharmac. Ther. 29:69-92 (1985)). These cells can then be delivered to the patient directly or after inclusion.

[0128] In some embodiments, nucleic acids are introduced into cells prior to in vivo administration of the resulting recombinant cells. In some embodiments, a technique can be used to provide stable introduction of nucleic acids into cells so that they are expressible by the cells and, in some cases, heritable and expressible by their offspring. Recombinant cells can be delivered to patients by various means. In some embodiments, the RdCVF protein is expressed from cells via a controllable, inducible, and / or repressible promoter.

[0129] In some embodiments, the cells used are autologous cells, allogeneic cells, or heterogeneous cells with respect to the patient. In some embodiments, autologous cells are manipulated ex vivo to contain the nucleic acid of the present invention, which enables the cells to produce or secrete the RdCVF protein, and then the cells are returned to the patient.

[0130] In some embodiments, cells are administered locally (e.g., intra-articular, intravitreous, intraretinal, intracranial, etc.) or systemically (e.g., IV).

[0131] In some embodiments, recombinant blood cells (e.g., hematopoietic stem cells and / or progenitor cells) are administered intravenously. In some embodiments, ophthalmic cells and / or pluripotent cells can be injected directly into the eye.

[0132] Stem cells and / or progenitor cells that can be isolated and maintained in vitro can potentially be used according to several embodiments of the present invention. Such stem cells include, but are not limited to, hematopoietic stem cells (HSCs), stem cells of epithelial tissues such as the skin and intestinal endometrium, embryonic cardiomyocytes, liver stem cells (see, e.g., WO94 / 08598), and neural stem cells (e.g., Stemple and Anderson (1992) Cell 71:973-985). In some embodiments, the cells administered are stem cells containing the nucleic acids of the present invention that can express and secrete RdCVF.

[0133] Encapsulated cells can enable controlled and / or continuous delivery of proteins such as RdCVF in vivo. In some embodiments, cells containing the nucleic acid of the present invention and expressing and / or secreting RdCVF are encapsulated. In some embodiments, the cells are encapsulated within a semipermeable membrane that allows diffusion of RdCVF across the membrane. Further information relating to encapsulated cells and encapsulated cell implants can be found in Sieving et al. (Proc Natl Acad Sci USA, (2006) 103(10):3896-901), U.S. Patent Nos. 7,115,257 and 7,820,195, and PCT Publication No. WO2011 / 044216. In some embodiments of the present invention, encapsulated cells expressing the RdCVF protein are delivered to animals.

[0134] In some embodiments, the inclusion cells are transplanted into a mammal (e.g., the eye, brain, or olfactory region). In some embodiments, the inclusion cells are retinal pigment epithelial cells (e.g., ARPE-19 (available from ATCC, Manassas, VA)). In some embodiments, the inclusion cells are used to deliver RdCVF to the eye (e.g., the fundus).

[0135] In some embodiments, the inlaid cell implant of the present invention comprises cells encapsulated in a compartment of a semipermeable hollow fiber membrane, the cells being genetically modified to produce RdCVF. In some embodiments, the inlaid cell implant has a suture loop at one end for fixing it to the sclera within the vitreous retina in the eye. In some embodiments, the inlaid cell implant is 3, 4, 5, 6, 7, 8, 9, or 10 mm in length.

[0136] RdCVF protein secretion and production The nucleic acids and viral vectors of the present invention can be used to express, produce, and / or secrete RdCVF from cells. This expression, production, and / or secretion can be induced in vitro, in vivo, or ex vivo.

[0137] Some embodiments of the present invention provide a method for causing cells to secrete the RdCVF protein, comprising administering the nucleic acid and / or viral vector of the present invention to the cells. In some embodiments, the cells may be mammalian cells, human cells, ophthalmic cells, retinal pigment epithelial (RPE) cells, rod cells, or cone cells.

[0138] Some embodiments of the present invention utilize vertebrate or mammalian cells. Examples of useful mammalian host cell lines include: SV40-transformed monkey kidney CVI cell line (e.g., COS-7, ATCC CRL 1651); human embryonic kidney cell line (e.g., 293 Freestyle (Invitrogen, Carlsbad, CA), including 293 or 293T cells, or 293FT cells, which are subcloned cell lines for growth in suspension culture (Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (e.g., BHK, ATCC CCL 10); Chinese hamster ovary cells (CHO cells); Chinese hamster ovary cells / -DHFR (e.g., CHO, Urlaub et al.) al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); Mouse Sertoli cells (e.g., TM4, Mather, Biol. Reprod. 23:243-251 (1980)); Monkey kidney cells (e.g., CVI ATCC CCL 70); African green monkey kidney cells (e.g., VERO-76, ATCC CRL-1587); Human cervical cancer cells (e.g., HELA, ATCC CCL2); Canine kidney cells (e.g., MOCK, ATCC CCL34); CF2TH cells; Buffalo rat hepatocytes (e.g., BRL 3A, ATCC CRL 1442); Human lung cells (e.g., W138, ATCC CCL 75); Human liver cells (e.g., Hep G2, HB 8065); Mouse mammary cancer tumor cells (e.g., MMT 060562, ATCC CCL51); TRI cells (Mather et al.) al., Annals NYAcad.Sci.383:44-68(1983)); MRC5 cells; ARPE-19 cells (ATCC) and FS4 cells.

[0139] In some embodiments, the cells are selected from the group consisting of 293 cells, CHO cells, PerC6 cells, Vero cells, BHK cells, HeLa cells, COS cells, MDCK cells, 3T3 cells, or WI38 cells.

[0140] Some embodiments of the present invention provide isolated cells containing the nucleic acid of the present invention. In some embodiments, the nucleic acid is incorporated into the cell genome / DNA.

[0141] The present invention also includes a method for producing an RdCVF protein, comprising culturing cells under conditions that enable the expression and secretion of the RdCVF protein, and isolating the RdCVF protein from the cell culture, wherein the cells contain the nucleic acid of the present invention that encodes the RdCVF protein and enables the expression of the RdCVF protein (e.g., secretion of the RdCVF protein). In some embodiments, the nucleic acid comprises a nucleotide sequence comprising the coding sequence for the RdCVF protein, and the RdCVF coding sequence comprises a re-encoded sequence. The RdCVF protein can be an RdCVF1 or RdCVF2 protein, or can be a long or short form. In some embodiments, these methods further comprise the purification of the RdCVF protein from the cells and / or culture supernatant.

[0142] Furthermore, the present invention includes an RdCVF protein expressed by cells from the nucleic acid of the present invention. The present invention also provides a secreted form of the RdCVF protein of the present invention and a composition containing the secreted RdCVF protein of the present invention.

[0143] In some embodiments, the RdCVF protein expressed from cells is purified to a purity of at least 90%, at least 93%, at least 95%, at least 98%, at least 99.5%, or at least 99.9% relative to the total protein.

[0144] Compositions, formulations and preparations Some embodiments of the present invention provide compositions, formulations, or preparations containing the nucleic acids of the present invention, the vectors of the present invention, the RdCVF proteins of the present invention, or any combination thereof, such as pharmaceutical compositions.

[0145] Pharmaceutical formulations (e.g., for injection) are generally, but not necessarily, biocompatible solutions of the active ingredient, including, for example, Hank's solution, Ringer's solution, or phosphate-buffered saline. In some embodiments, the formulation or pharmaceutical composition comprises one or more of the following: citrate, NaCl, potassium chloride (KCl), calcium chloride dihydrate (CaCl2·2H2O), magnesium chloride hexahydrate (MgCl2·6H2O), sodium acetate trihydrate (CH3CO2Na·3H2O), sodium citrate dihydrate (C6H5O7Na3·2H2O), sucrose, sodium hydroxide and / or hydrochloric acid (to adjust pH), and water. The above list includes several molecules enumerated as specific hydrates (e.g., dihydrates, trihydrates, hexahydrates, etc.). Various hydrates of these compounds can be used in the present invention, and it is understood that the present invention is not limited to these specific hydrate forms of the enumerated molecules. In some embodiments, the formulation or pharmaceutical composition comprises one or more components selected from the group consisting of histidine, MgCl2, trehalose, polysorbate, polysorbate 20, NaCl, sucrose, arginine, and proline. In some embodiments, the formulation comprises one or more of histidine, α,α-trehalose dehydrated, MgCl2, polysorbate such as polysorbate 20, and NaCl. In some embodiments, the formulation or pharmaceutical composition comprises one or more of phosphate-buffered saline (PBS) and pluronic F-68. In some embodiments, the concentration of pluronic F-68 can be 0.0001%, 0.001%, 0.005%, 0.01%, or 0.1%.

[0146] Examples of formulations and methods of formulation suitable for the desired administration method can be found in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton, PA, and U.S. 7,208,577.

[0147] In some embodiments, compositions for in vivo use contain a “carrier” or “pharmaceutically acceptable carrier.” The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle administered together with the nucleic acid, vector, or protein of the present invention. The term “carrier” includes, but is not limited to, any inorganic or organic solid or liquid material, which may be synthetic or naturally derived, that is mixed or formulated together with the active ingredient of the composition to facilitate administration to a subject. Any other material conventionally used in the formulation of pharmaceuticals is also appropriate. In embodiments, the pharmaceutical carrier differs from typical solutions and suspensions in that it is specially prepared for in vivo use to eliminate substances that may be harmful to the host to which the composition is administered (e.g., for the removal of bacterial toxins).

[0148] Examples of suitable liquid carriers include water and aqueous solutions containing oxygen-containing organic compounds such as ethanol. Buffers and other materials commonly found in pharmaceutical preparations, such as flavorings and suspensions, may also be present. Generally, suitable oils, saline solutions, aqueous dextrose solutions (glucose), and related sugar solutions, as well as glycols such as propylene glycol or polyethylene glycol, are typically suitable carriers for parenteral solutions. In some embodiments, the solution for parenteral administration contains a water-soluble salt of the active ingredient, a suitable stabilizer, and a buffering agent if desired or necessary. Antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid can be used as stabilizers, either alone or in combination. Citric acid and its salts, as well as sodium EDTA, are also used. In addition, the parenteral solution may contain preservatives such as benzalkonium chloride, methyl or propylparaben, and chlorobutanol.

[0149] In embodiments, the carrier is a carbohydrate and includes, but is not limited to, trehalose, mannitol, glutathione, xylitol, sucrose, lactose, and sorbitol. In embodiments, the formulations of the present disclosure include natural or synthetic surfactants, such as DPPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). In embodiments, the formulations of the present invention include polyethylene glycol. In embodiments, the formulations of the present invention include a dextran such as cyclodextran. In embodiments, the formulations of the present invention include a cyclodextrin, a tertiary amine, and / or a beta-cyclodextrin. In embodiments, the formulation of the present invention comprises an enhancer such as a bile salt. In embodiments, the formulation of the present invention comprises cellulose and cellulose derivatives. In embodiments, the formulation of the present invention comprises amino acids. In embodiments, the formulation of the present invention is intended to comprise liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers.

[0150] In embodiments, the formulation of the present invention comprises a wetting agent and / or an emulsifier, and / or a pH buffer. In embodiments, the formulation of the present invention comprises a solubilizer, and / or a local anesthetic such as lignocaine for relieving pain at the injection site.

[0151] In some embodiments, the pharmaceutical preparation or composition of the present invention comprises (i) a pharmaceutically acceptable carrier and (ii) the nucleic acid of the present invention, the viral vector of the present invention, the RdCVF protein of the present invention, or any combination thereof.

[0152] Treatment, administration, and delivery RdCVF proteins have been shown to promote the survival of cone photoreceptor cells in vitro and in vivo. For example, intraocular injection of a short form of human RdCVF1 (RdCVF1S) protein not only rescued cone cells from degeneration but also preserved their function in an animal model of hereditary retinal degeneration (Yang et al. (Mol Therapy (2009) 17:787-795 and supplemental materials)). Endogenous RdCVF1 expression is mainly limited to the retina (Leveillard et al. (2004) Nature Genetics 36:755-759).

[0153] In embodiments, the present disclosure provides a method for preserving rod and / or cone cells, comprising administering to the eye of a mammal the nucleic acid of the present invention, the vector of the present invention (e.g., a viral vector), the RdCVF protein of the present invention, the pharmaceutical composition of the present invention, or a combination thereof. In embodiments, the term “preservation” means maintaining the function of the rod cells, for example, the function being maintained at 100%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the function of healthy rod cells. In some embodiments, the viral vector and / or nucleic acid of the present invention is administered by subretinal injection, intravitreous injection, injection into the anterior chamber of the eye, subconjunctival injection, sub-Tenon's capsule injection, or any combination thereof. In some embodiments, the mammal to be treated is a domesticated mammal, such as a cat, dog, or horse. In one embodiment, the mammal being treated is a human.

[0154] In embodiments, the present disclosure provides a method for preserving rod and / or cone cells, comprising administering to the eye of a mammal the nucleic acid of the present invention, the viral vector of the present invention, the RdCVF protein of the present invention, the pharmaceutical composition of the present invention, or a combination thereof. In embodiments, the term “preservation” means maintaining the function of the rod and / or cone cells, for example, the function being maintained at 100%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the function of a healthy cone cell. In some embodiments, the viral vector and / or nucleic acid of the present invention is administered by subretinal injection, intravitreous injection, injection into the anterior chamber of the eye, subconjunctival injection, sub-Tenon’s capsule injection, or any combination thereof. In some embodiments, the mammal to be treated is a domesticated mammal, such as a cat, dog, and horse. In embodiments, the mammal to be treated is a human.

[0155] In some embodiments, the treated mammals suffer from eye diseases such as retinal dystrophy, Stargardt disease, retinitis pigmentosa, dry age-related macular degeneration (dry AMD), geographic atrophy (an advanced stage of dry AMD), wet age-related macular degeneration (wet AMD), glaucoma / ocular hypertension, diabetic retinopathy, Valday-Biedl syndrome, Bassen-Kohnzweig syndrome, Best's disease, choroidopathy, gynostosis, congenital amaurosis, Levsan syndrome, Usher syndrome, thyroid-related eye diseases, Graves' disease, diseases associated with retinal pigment epithelial cells, anterior segment diseases, lens diseases / cataracts, eyecup syndrome, or uveitis. In some embodiments, the preserved rod cells do not contain the nucleic acids and / or viral vectors of the present invention. For example, the preserved ophthalmic cells are not preserved by transduction of the preserved ophthalmic cells themselves.

[0156] This disclosure further provides a method for maintaining the function of rod and / or cone cells of an eye, comprising administering the nucleic acid and / or viral vector of the present invention to the eye of a mammal, wherein the nucleic acid and / or viral vector is administered by subretinal injection, and the rod and / or cone cells are preserved at a location at least 1 mm, at least 2 mm, at least 3 mm, at least 5 mm, at least 7 mm, at least 10 mm, at least 15 mm, at least 20 mm, at least 25 mm, or at least 2 cm from the subretinal injection site. For example, although we do not wish to be bound by theory, cells transduced with the nucleic acid or viral vector at the subretinal injection site may express and / or secrete RdCVF-long protein, which can provide a preservation effect on the rod and / or cone cells of the eye at a location far from the transduced cells or the injection site.

[0157] If the introduction or administration of nucleic acids or vectors encoding the RdCVF protein is disclosed, the disclosure is understood to also provide the introduction or administration of the RdCVF protein itself. If the introduction of the RdCVF protein is disclosed, the present invention is understood to also disclose the introduction of nucleic acids or vectors encoding the RdCVF protein.

[0158] In some embodiments, the compositions of the present invention are administered topically or systemically. Useful routes of administration are described herein and are well known in the art. Methods of introduction or administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intratracheal, topical, inhalation, percutaneous, rectal, parenteral routes, epidural, intracranial, intracerebral, intraventricular, subdural, intraarticular, intrathecal, intracardiac, intracoronal, intravitreous, subretinal, intrachorionic, subconjunctival, sub-Tenon's capsule injection, eye drops, oral routes, via balloon catheter, via stent, or any combination thereof. Systemic administration may be by intravenous or intra-arterial injection, or by transmucosal, subcutaneous, percutaneous, and / or intraperitoneal delivery.

[0159] In some embodiments, for example, in embodiments involving administration to the eye, the RdCVF1L or RdCVF2L coding vector or nucleic acid of the present invention is administered approximately once every week, month, two months, three months, six months, nine months, one year, 18 months, two years, 30 months, three years, five years, or ten years, or as needed. In some embodiments, for example, in embodiments involving administration to the eye, the RdCVF coding vector or nucleic acid of the present invention is administered approximately every 1 - 4 weeks, every 4 - 8 weeks, every 1 - 4 months, every 3 - 6 months, every 4 - 8 months, every 6 - 12 months, every 9 - 15 months, every 12 - 18 months, every 15 - 21 months, every 18 - 24 months, every 1 - 2 years, every 1.5 - 3 years, every 2 - 4 years, every 3 - 5 years, every 5 - 7 years, every 7 - 10 years, or every 10 - 20 years. Administration of the vector encoding the RdCVF protein is expected to be less frequent than administration of the RdCVF protein itself. In some embodiments of the present invention, the pharmaceutical preparation contains the vector encoding the RdCVF protein of the present invention, and the pharmaceutical preparation is administered to the patient only once.

[0160] <00,00628>In some embodiments, the RdCVF1L or RdCVF2L coding vector or nucleic acid of the present invention is administered to the human eye by intravitreal injection or subretinal injection. In some embodiments, about 15 μg to about 5 mg, about 15 μg to about 500 μg, about 100 μg to about 900 μg, about 300 μg to about 700 μg, about 500 μg to about 1 mg, about 1 mg to about 5 mg, about 1 mg, or about 500 μg of the RdCVF protein is administered to the human eye by intravitreal injection or subretinal injection.

[0161] In some embodiments, the RdCVF1L or RdCVF2L coding vector or nucleic acid of the present invention is administered by subretinal injection or intravitreal injection. In some embodiments, about 5×10 8 ~ about 1×10 9 、 about 5×10 8 ~ about 7.5×10 8 、 about 7.5×10 8 ~ about 1×10 9 、 about 6×108 ~Approx. 9×10 8 , about 7×10 8 ~Approx. 8×10 8 , about 5×10 8 , about 6×10 8 , about 7×10 8 , about 8×10 8 , about 9×10 8 , or approximately 1 x 10 9 , or approximately 1 x 10 10 , or approximately 1 x 10 11 , or approximately 1 x 10 12 AAV vectors with a number of vector genome copies (GCs) are administered by subretinal injection. In some embodiments, the AAV vector has approximately 5 × 10¹⁶ 8 ~Approx. 1×10 10 , about 5×10 8 ~Approx. 5×10 9 , about 5×10 8 ~about 2×10 9 , about 2×10 9 ~Approx. 5×10 9 , about 5×10 9 ~Approx. 1×10 10 , about 5×10 8 ~Approx. 1×10 9 , about 1×10 9 ~Approx. 3×10 9 , about 3×10 9 ~about 6×10 9 , about 6×10 9 ~Approx. 1×10 10 , about 1×10 9 ~Approx. 1×10 10 , about 1×10 10 ~Approx. 1×10 11 , or 1 × 10 11 ~Approx. 1×10 12 , or 1 × 10 12 ~Approx. 5×10 12 Each GC is administered by intravitreal injection. In some embodiments, approximately 5 × 10⁶ AAV vectors are used. 8 ~Approx. 1×10 10 , about 5×10 8 ~Approx. 5×10 9 , about 5×10 8 ~about 2×10 9 , about 2×10 9 ~Approx. 5×10 9, about 5×10 9 ~ about 1×10 10 , about 5×10 8 ~ about 1×10 9 , about 1×10 9 ~ about 3×10 9 , about 3×10 9 ~ about 6×10 9 , about 6×10 9 ~ about 1×10 10 , about 1×10 9 ~ about 1×10 10 , about 1×10 10 ~1×10 11 , 1×10 11 ~ about 1×10 12 , 1×10 12 ~ about 1×10 13 , 1×10 13 ~ about 1×10 14 , 1×10 14 ~ about 5×10 14 of GC are administered by intrathecal injection. In some embodiments, about 5×10 8 ~ about 1×10 10 , about 5×10 8 ~ about 5×10 9 , about 5×10 8 ~ about 2×10 9 , about 2×10 9 ~ about 5×10 9 , about 5×10 9 ~ about 1×10 10 , about 5×10 8 ~ about 1×10 9 , about 1×10 9 ~ about 3×10 9 , about 3×10 9 ~ about 6×10 9 , about 6×10​​​​​​​​​​​​​​​​​​​​​​14 , 1 x 10 14 ~Approx. 1×10 15 The GCs are administered by intravenous injection. It is understood that the amount of AAV vector may be measured in terms of transduction units or GC count. The GC count is typically 25 to 300 times higher than when the same AAV vector sample is measured for transduction units.

[0162] This disclosure provides a method for treating a disease comprising administering to a mammal a nucleic acid, a viral vector, an RdCVF protein, a pharmaceutical composition, or a combination thereof, of the present invention, the disease being a central nervous system (CNS) disease. In some embodiments, the CNS diseases to be treated are Alzheimer's disease, Huntington's disease, Parkinson's disease, and olfactory disorders. In some embodiments, the viral vector of the present invention is an AAV vector.

[0163] Alzheimer's disease (AD) is the most common age-related neurodegenerative disease and the leading form of dementia, affecting approximately 50 million people worldwide. The number of AD patients is projected to triple by 2050. The disease primarily affects patients over 65 years of age and is characterized by a gradual decline in cognitive function. It is a progressive disease that can begin with mild memory loss and lead to a loss of the ability to continue conversations and cope with daily life. Currently, there is no cure for AD, but immunotherapy may improve symptoms or slow its progression.

[0164] Pathologically, this disease is characterized by the accumulation of extracellular β-amyloid (Aβ) peptide plaques and intracellular neurofibrillary entanglement (NFT). The 40- or 42-amino acid Aβ peptide is produced by the proteolytic cleavage of amyloid precursor protein (APP), while NFT consists of highly phosphorylated and misfolded tau protein. These neuropathological features are accompanied by severe neuroinflammation characterized by the activation of stellate cells and microglia. The neurotoxicity of Aβ and NFT in the brain is generally considered to be the cause of synaptic dysfunction and neurodegeneration. Mutations in APP or tau cause cognitive impairment and / or neuronal loss in mouse models of AD.

[0165] Neurotrophic factor therapy is a neuroprotective approach in the AD brain, potentially slowing disease progression by preventing neurodegeneration and stimulating neuronal function in Alzheimer's disease, as an alternative to amyloid-modifying agents. Administration of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) has shown improvements in learning and memory, as well as prevention of neuronal cell death, in mouse models of AD. Gene therapy offers a unique approach to treating this chronic disease, as it allows for the sustained expression of these factors in the brain. Indeed, gene therapies for AD using AAV to deliver NGF or BDNF are currently in clinical trials.

[0166] Rod and cone photoreceptors are specialized nerve cells that function in the early stages of vision (Molday and Moritz, J Cell Sci., 128:4039 (2015)). The RdCVF protein possesses neuroprotective activity and is a factor for cone and / or rod survival, as well as a general neuronal survival factor. The fact that RdCVF administration preserves rod and cone function, as well as the success of in vitro studies using human neuroblastoma cell lines and human neural progenitor cells disclosed herein, predicts the neuroprotective effects of RdCVF administration. Therefore, the disclosure herein provides a method for protecting brain nerve cells, which is beneficial for the treatment of CNS diseases such as Alzheimer's disease.

[0167] Endogenous RdCVF1 expression is mainly limited to the outer nuclear layer containing rod and cone photoreceptor cells, and endogenous RdCVF expression could not be detected in other organs / tissues such as the kidney, testes, spleen, intestine, lung, and cerebellum (Leveillard, T. (2004) Nature Genetics, 36:755-759). Using a proteomics approach, 90 proteins, including the microtubule-associated protein tau, were found to interact with RdCVF (Fridlich et al. Mol Cell Proteomics (2009) 8(6):1206-1218). Fridlich et al. demonstrated that tau phosphorylation levels are increased in the retina of Nxnl1- / -(RdCVF1- / -) mice, possibly due to oxidative stress in some cases, as tau is hyperphosphorylated in the brains of patients with Alzheimer's disease. Furthermore, Fridlich et al. showed that RdCVFL inhibits tau phosphorylation. Cronin et al. (Cell Death and Differentiation (2010) 17:1199-1210) found that the Nxnl1- / -(RdCVF1- / -) retina contains aggregated tau protein, as seen in the brains of patients with Alzheimer's disease.

[0168] Mice lacking RdCVF2 have impaired vision and smell. Normal mice express RdCVF2 in their olfactory epithelium. Jaillard et al. (ARVO meeting (2009) program # / poster #491 / D636) reported that olfactory neurons survived at a higher rate when cultured in the presence of RdCVF2. Jaillard et al. also compared RdCVF2- / - mice to control mice by performing an olfactory discrimination learning test. By 12 months of age, RdCVF2- / - mice no longer responded correctly to stimuli.

[0169] Therefore, based on the above, the RdCVF coding nucleic acid, viral vector, or RdCVF protein of the present invention can be used to treat or improve Alzheimer's disease, Huntington's disease, Parkinson's disease, and olfactory disorders.

[0170] All publications, patents, patent applications, and GenBank sequences described herein are incorporated herein by reference to the same extent as any individual publication, patent, or patent application is specifically and individually indicated as being incorporated herein by reference. Any supplemental information published in conjunction with any of the aforementioned publications, patents, and patent applications is also incorporated by reference. For example, some journal articles are published with supplemental information that is typically available online.

[0171] While specific embodiments of the present invention are described herein for illustrative purposes, those skilled in the art will understand that numerous modifications of the details can be made without departing from the present invention as set forth in the appended claims.

[0172] array Sequence ID 1 - pAAV.hIgK.hRdCVF1L shuttle vector sequence for mammalian expression cassette of human RdCVF1L (-Met) and human IgK signaling sequence (+Asp) into rAAV. [Sequence Listing 1-1] JPEG2026516412000001.jpg113170 [Sequence Listing 1-2] JPEG2026516412000002.jpg218170 [Sequence Listing 1-3] JPEG2026516412000003.jpg25170 Sequence ID 2-rAAV2.hIgK.hRdCVF1L Vector Genome DNA Sequence [Sequence Listing 2-1] JPEG2026516412000004.jpg185170 [Sequence Listing 2-2] JPEG2026516412000005.jpg19170 SEQ ID NO: 3 - Amino acid sequences of human RdCVF1L (-Met) and human IgK signaling sequence (+Asp) [Sequence Listing 3] JPEG2026516412000006.jpg24170 SEQ ID NO: 4 - Amino acid sequence of human RdCVF1L(-Met) and human IgK signaling sequence [Sequence Listing 4] JPEG2026516412000007.jpg22170 Sequence ID 5 - Amino acid sequences of human RdCVF1S(-Met) and human IgK signaling sequence(+Asp) [Sequence Listing 5] JPEG2026516412000008.jpg19170 Sequence ID 6 - Amino acid sequence of human RdCVF1S(-Met) and human IgK signaling sequence [Sequence Listing 6] JPEG2026516412000009.jpg17170 Sequence ID 7 - Amino acid sequence of human IgK signaling sequence (+Asp) [Sequence Listing 7] JPEG2026516412000010.jpg9170 Sequence ID 8 - Amino acid sequence of the human IgK signaling sequence [Sequence Listing 8] JPEG2026516412000011.jpg9170 Sequence ID 9 - Amino acid sequence of human IgK signaling sequence I [Sequence Listing 9] JPEG2026516412000012.jpg10170 Sequence ID 10 - Nucleotide sequences of human RdCVF1L(-Met) and human IgK signal sequence I [Sequence Listing 10-1] JPEG2026516412000013.jpg13170 [Sequence Listing 10-2] JPEG2026516412000014.jpg191170 SEQ ID NO: 11-Human RdCVF1L(-Met) and Human IgK Signal Sequence I Amino Acid Sequence [Sequence List 11] JPEG2026516412000015.jpg23170 Nucleotide sequence of sequence number 12-RdCVF2L [Sequence Listing 12] JPEG2026516412000016.jpg39170 Amino acid sequence of SEQ ID NO: 13-RdCVF2L [Sequence List 13] JPEG2026516412000017.jpg17170 Codon-optimized nucleotide sequences of sequence number 14-RdCVF2L(-Met) and human IgK signaling sequence (+Asp) [Sequence Listing 14] JPEG2026516412000018.jpg42170 Amino acid sequences of sequence number 15-RdCVF2L(-Met) and human IgK signaling sequence (+Asp) [Sequence Listing 15] JPEG2026516412000019.jpg20170 [Examples]

[0173] The present invention will now be described with reference to the following examples. These examples are provided for illustrative purposes only, and the present invention should not be construed as being limited to these examples, but rather as encompassing all possible modifications that become apparent as a result of the teachings provided herein.

[0174] Example 1 - Expression of human rod-derived pyramidal survival factor-length containing human immunoglobulin light chain signaling peptide mediated by recombinant adeno-associated virus. Plasmid cloning The nucleotide sequence of human L1 IgK light chain leader sequence (hIgK) + aspartic acid (D or Asp) was added to the N-terminus of a codon-optimized human (RdCVF1L) open reading frame by PCR amplification from the in-house plasmid pAAV-LRr268 ​​for the production of an AAV vector encoding the protein of SEQ ID NO: 3 (RdCVF1L and the human IgK signal sequence of SEQ ID NO: 7). The 737 bp hIgK.hRdCVF1L PCR product and pAAV-LRr268 ​​were digested with EcoR1 and Nhe1, and the hIgK.hRdCVF1L fragment was ligated to the 4627 bp fragment of pAAV-LRr268 ​​to create the plasmid pAAV.hIgK.hRdCVF1L (SEQ ID NO: 1). Sequence identity of pAAV.hIgK.hRdCVF1L was confirmed by Sanger sequencing.

[0175] Production and purification of rAAV2.hIgK.hRdCVF1L (SEQ ID NO: 2) HEK293FT cells were cultured in DMEM containing GlutaMAX and supplemented with non-essential amino acids, sodium pyruvate, and 10% FBS and antibiotics (100 μg / mL penicillin and 50 μg / mL streptomycin). Approximately 72 hours before transfection, cells were counted and seeded into 18 15 cm dishes at a rate of 1.5 × 10⁶ cells per dish. On the day of transfection, the growth medium was removed and replaced with fresh medium. The cells were then transfected with PEI (1 μg / μL) using a PEI:DNA mass ratio of 2. Each 15 cm dish was transfected with pRC2c, pHelper in a molar ratio of 1:1:1, and 50.75 μg (0.35 μg / cm²) of plasmid DNA in the rAAV2.hIgK.hRdCVF1L vector. To prepare the PEI-DNA complex, the plasmid was diluted in DMEM (2 mL / 15 cm dish, no additives) and then diluted with PEI. This solution was mixed and incubated at room temperature for 20 minutes, then gently mixed again before application to cells. After approximately 72 hours, the cells were scraped, and the cells and medium were collected and centrifuged at 1000 × g for 5 minutes at 4°C. After centrifugation, the medium was decanted, and the virus present in the medium was recovered by PEG precipitation by adding 5 g of PEG8000 and 0.3 g of NaCl to each 50 mL of medium and incubating the medium overnight at 4°C. The cell pellet was resuspended in lysis buffer (dPBS + 0.01% pluronic F68 and 200 mM NaCl) and lysed by sonication following one freeze-thaw cycle. The lysate was clarified by centrifugation at 3000 × g for 30 minutes at 4°C, and the supernatant was collected. After incubation, the PEG precipitate was centrifuged at 3000 × g for 30 minutes at 4°C, and the medium was discarded. The PEG pellet was resuspended in lysis buffer and combined with the clarified cell lysates, and incubated with benzonase (50 U / mL) at 37°C for 60 minutes. The lysates were then ultracentrifuged through an IDX (iodixanol) density gradient.IDX was diluted in dPBS and laid in 38.5 mL quick-seal tabs in the following order: 8 mL of 15% IDX, 6 mL of 25% IDX, 8 mL of 40% IDX, and 5 mL of 60% IDX, all containing 1 M NaCl. The clarified lysates were placed on top of the IDX gradient and centrifuged in a T70i rotor at 60,000 RPM (264,904 × g) at 10°C for 180 minutes. After centrifugation, the 3 mL 40% IDX layer was collected by puncturing an ultracentrifugation tube with an 18 g needle just below the 40–60% IDX interface. The collected IDX containing AAV particles was buffer-changed by repeated dilution with formulation buffer (dPBS containing 0.001% pluronic F68) and concentration through an Amicon Ultra-15 100K centrifuge filter. After the final concentration, the AAV samples were sterilized by centrifugation through a 0.22 μM filter, divided into equal portions, and stored at -80°C.

[0176] qPCR analysis for genome copy titer determination The purified rAAV2.hIgK.hRdCVF1L titer, defined as genome copy (GC) / mL, was determined by qPCR using the standard curve method. A standard curve for calculating genome copy titer was generated by linearizing the pAAV.EGFP control vector with HindIII. The concentration of the gel-purified linearized vector was determined by spectrophotometric analysis, and 2 × 10⁻⁶ values ​​were obtained. 8 From 2 x 10 3The samples were serially diluted to copies / μL. Before performing qPCR, rAAV samples were digested with DNase I to degrade any contaminated pAAV.hIgK.hRdCVF1L transfer vectors. 10 μL of rAAV sample was diluted in 79 μL of nuclease-free H2O, 10 μL of DNase reaction buffer, and 1 μL of DNase I, and incubated at 37°C for 20 minutes and at 75°C for 10 minutes. DNase I-digested samples were serially diluted in 10 mM TRIS-HCl (1:10 and 1:100) and stored on ice for qPCR. Three 5 μL replicates of each sample dilution and standard curve concentration were added to 15 μL of iQ SYBR Green Supermix containing 0.67 μM FWD and REV primers targeting the CMV promoter of the rAAV transgene expression cassette. qPCR was performed on a Bio-RAD iCycle thermocycler using the following protocol: 40 cycles of 3 minutes at 98°C (15 seconds lysis at 98°C, 30 seconds annealing / extension at 58°C). A melting curve from 55°C to 98°C was performed to verify the specificity of PCR amplification. Following PCR, the amplification curve was (automatically) subtracted from the baseline to determine the CT value, defined as the number of cycles to reach the threshold, for each standard and unknown sample. The standard curve was plotted using plasmid standards fitted by linear regression to determine the concentration of the rAAV2.hIgK.hRdCVF1L dilution. The titer was calculated according to the following formula: Titer (GC / mL) = Volume (calculated relative to the standard curve) × 10 (DNase I dilution) × 2 (double-stranded DNA standard) × Dilution factor × 1000 (convert μL to mL). The titer of rAAV2.hIgK.hRdCVF1L is 9.94 × 10 12 The result was GC / mL.

[0177] Silver staining analysis of rAAV2.hIgK.hRdCVF1L 5 x 10 9Aliquotes of rAAV2.hIgK.hRdCVF1L containing 1 genome copy were denatured by reducing the sample buffer at 95°C for 5 minutes and then electrophoresed on a 4-15% polyacrylamide gel. The gel was then stained using the Pierce silver staining kit according to the manufacturer's instructions. Briefly, the gel was washed in ultra-high purity H2O for 2 × 5 minutes and fixed in 30% ethanol with 10% acetic acid for 2 × 15 minutes. After fixation, the gel was washed in 10% ethanol for 2 × 5 minutes, then in ultra-high purity H2O for 2 × 5 minutes. The gel was then incubated in sensitizer solution (50 μL of sensitizer in 25 mL of ultra-high purity H2O) for 1 minute and washed in ultra-high purity H2O for 2 × 1 minute. Next, the gel was stained for 30 minutes (0.5 mL of enhancer in 25 mL of staining solution) and washed in ultra-high purity H2O for 2 × 20 seconds. For development, the gel was immersed in a developer standard solution (0.5 mL enhancer in 25 mL of developer) until protein bands appeared. Gel development was stopped after 10 minutes with 5% acetic acid. The gel was washed with ultra-high purity H2O and imaged using an Epson V700 photoscanner. The presence of AAV particles was confirmed by visualization of VP1, VP2, and VP3 capsid proteins (Figure 1).

[0178] In vitro assay of rAAV2.hIgK.hRdCVF1L gene transfection Protein expression and hRdCVF1L secretion after transduction of rAAV2.hIgK.hRdCVF1L were evaluated in human retinal pigment epithelial cell line APRE-19. ARPE-19 cells were placed in a 6-well plate at a rate of 2 × 10⁶ cells per well. 5Cells were plated in 1 mL of complete DMEM and incubated overnight. Cells were then transduced with either rAAV2.hIgK.hRdCVF1L or rAAV2.EGFP control vector at a MOI of 1,000. After 48 hours of incubation, the medium was collected, centrifuged at 16,000×g for 30 minutes at 4°C, and stored at -20°C. Cells were harvested in dPBS- / - supplemented with 10 mM EDTA. Cells were centrifuged at 500×g for 5 minutes, and the cell pellet was lysed in 75 μL of ice-cold RIPA buffer supplemented with a Halt protease inhibitor cocktail. Cell lysates were incubated on ice for 30 minutes, centrifuged at 16,000×g for 30 minutes at 4°C, and stored at -20°C.

[0179] For Western blotting, the culture medium samples and cell lysates were thawed on ice. 5 microliters of each cell lysate were added to 11 μL of dPBS using 4 μL of 5x reducing sample buffer and denatured at 95°C for 5 minutes. For the culture medium samples, 16 μL was added to 4 μL of 5x reducing buffer and denatured at 95°C for 5 minutes. The samples were electrophoresed on 4-15% polyacrylamide gel and blotted onto nitrocellulose membranes. The nitrocellulose membranes were blocked in 1× casein at room temperature for 1 hour and incubated overnight at 4°C with primary rabbit anti-RdCVF1L (AD-10, 1:1000) antibody in 1× casein. After the primary incubation, the membranes were washed with 1× casein for 3×5 minutes and incubated with secondary biotinylated goat anti-rabbit antibody (1:5,000) in 1× casein at room temperature for 1 hour. After secondary incubation, the membrane was washed in 1x casein for 3x5 minutes and incubated in Vectastain ABC-AMP (30 μL A and 30 μL B in 1x casein for 15 minutes) at room temperature for 40 minutes. Next, the membrane was washed in 0.1 M Tris-HCl (pH 9.5) for 3x5 minutes, incubated in Duolax for 5 minutes, and washed again in 0.1 M Tris-HCl for 5 minutes. The membrane was exposed to BioMax optical film for 180 seconds, and then the film was immersed in developer solution (26 mL of developer and replenisher solution in 92 mL of H2O) for 1 minute and fixer solution (26 mL of fixer and replenisher solution in 92 mL of H2O) for 1 minute. The film was then rinsed with H2O, dried, and imaged using an Epson V700 photoscanner. A protein duplex immunoreactive to the anti-RdCVF1L antibody was detected at approximately 30 kDa (close to the theoretical molecular weight of hRdCVF1L) in cell lysates and culture media from ARPE-19 cell cultures transduced with rAAV2.hIgK.hRdCVF1L, but was not detected with the control vector rAAV2.EGFP (Figure 2).

[0180] Example 2 - Expression and secretion of human RdCVF1L mediated by rAAV vectors, compared with different human IgK signaling sequences.

[0181] A recombinant AAV vector encoding the human IgK signal sequence (+Asp) (SEQ ID NO: 7) and human RdCVF1L was described in Example 1. A recombinant AAV vector (SEQ ID NO: 8) encoding the protein of SEQ ID NO: 4, which contains the human IgK signal sequence (-Asp), and the same human RdCVF1L was generated and named rAAV2.hIgK(-D)hRdCVF1L. The only difference between the two vectors (rAAV2.hIgK.hRdCVF1L and rAAV2.hIgK(-D).hRdCVF1L) was one extra amino acid, namely aspartic acid, in the signal sequence of the rAAV2.hIgK.hRdCVF1L vector. The two vectors were prepared and titrated in the same manner. The same number of human HEK293FT cells were transduced using the same amounts of the two vectors. 72 hours after transduction, the cell lysates and cell culture medium were treated as described above in Example 1 for RdCVF1L expression and secretion by Western blotting analysis. As shown in Figure 3, human RdCVF1L expression and secretion were far more efficient with the rAAV2.hIgK.hRdCVF1L vector compared to the rAAV2.hIgK(-D).hRdCVF1L vector. Surprisingly, the data suggest that even a difference of a single amino acid in the signal sequence can have a significant impact on protein expression and secretion (Figure 3).

[0182] Example 3 - RdCVF and Alzheimer's disease The trophic factor rod-derived cone survival factor (RdCVF) was initially identified by screening a mouse neuroretinal cDNA expression library for specific neuronal-rod photoreceptor cell rescue. RdCVF has two isoforms, namely truncated (RdCVFS) and full-length (RdCVFL), resulting from alternative splicing of the nucleoledoxin-like (nxnl1) gene. Knockout of the nxnl1 gene induces degeneration of mouse photoreceptors, suggesting that at the gene level, this gene is essential for maintaining the survival of mouse photoreceptors. We found that intraocular administration of RdCVF1L mediated by adeno-associated virus (AAV) significantly increased photoreceptor survival in a mouse model of hereditary retinal degeneration (US9,265,813 and Figure 4).

[0183] The AAV2-RdCVF1L vector was injected into the subretinal space of one eye of rd10 mice at 3 days postnatal, while the other eye was left to serve as a control. In this clinically relevant model of retinal degeneration, mice were sacrificed at 10 weeks of age, when retinal photoreceptor cells completely degenerate without any treatment. The eyes were excised and further processed for histological analysis. Light microscopy showed that approximately five layers of photoreceptor nuclei remained in the upper hemisphere of the vector-injected eye (Figure 4, left panel). In contrast, the untreated contralateral eye had no or very few photoreceptor nuclei remaining in the retina (Figure 4, right panel).

[0184] As described above, we demonstrated the neuroprotective effect of RdCVF1L in retinal neurons. Since the retina is an extension of the brain located behind the eye, we hypothesized that similar protection by RdCVF1L could be applied to brain neurons. Therefore, to test this hypothesis, we selected two commercially available human neuronal cell lines (human neuroblastoma and human neural progenitor cells). Surprisingly, we found that RdCVF1L could protect these neurons from β-amyloid-induced toxicity (Figures 5 and 6).

[0185] The human neuroblastoma cell line SH-SY5Y was purchased from ATCC (Manassas, VA) and used in a lactate dehydrogenase (LDH) cytotoxicity assay. To measure the release of LDH from damaged cells, an LDH assay was performed using the CytoTox 96® non-radioactive cytotoxicity assay kit (Promega).

[0186] In short, 1 × 10⁶ SH-SY5Y cells were seeded overnight in a 6-well plate. The following day, the standard DMEM medium was removed, and the cells were treated for 24 hours in 500 μL of serum-free DMEM medium containing 1L of 175 or 350 ng / mL of RdCVF. Subsequently, Aβ1-42 (0, 2.5, 5, and 10 μM, Invitrogen) was added to each well for 24 hours. 50 μL of the medium supernatant and cell lysates were used for the LDH assay. Cytotoxicity was calculated against control cells. 2.5 and 5 μM Aβ did not cause significant toxicity compared to the vehicle, but 10 μM caused a significant increase in LDH release (P<0.001). Surprisingly, treatment with RdCVF1L at both concentrations significantly reduced LDH release induced by 10 μM Aβ compared to vehicle treatment (P<0.001), demonstrating the neuroprotective effect of RdCVF1L (Figure 5).

[0187] To further confirm RdCVF1L's effect on Aβ neurotoxicity, similar experiments were performed using human neural progenitor (NHNP) cells (Lonza, Walkersville, MD). NHNP cells were cultured and further differentiated into neurons using the Neural Progenitor Differentiation Medium Bullet Kit (Lonza), which contains the necessary additives and medium for optimal NHNP differentiation. Differentiated NHNP cells were cultured for 8 days, incubated for 24 hours with 100 and 200 ng / mL of RdCVF1L or vehicle, and then toxicized with 10 μM Aβ for 2 days. LDH release was measured in the culture supernatant and cell lysates. Again, surprisingly, RdCVF1L was found to significantly reduce Aβ-induced LDH release compared to vehicle, even at lower concentrations (P<0.001), further supporting its neuroprotective effect (Figure 6).

Claims

1. A nucleic acid comprising a nucleotide sequence encoding a full rod-derived cone survival factor ("RdCVF-long" or "RdCVFL") protein and a nucleotide sequence encoding a human immunoglobulin kappa chain (IgK) signal sequence, wherein the human IgK signal sequence comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

7.

2. The nucleic acid according to claim 1, wherein the RdCVFL protein is RdCVF1L protein or RDCVF2L protein.

3. The nucleic acid according to claim 1 or 2, wherein the RdCVFL protein is the RdCVF1L protein.

4. The nucleic acid according to any one of claims 1 to 3, wherein the RdCVFL protein is human RdCVFL protein.

5. The nucleic acid according to any one of claims 1 to 4, wherein the nucleotide sequence encoding the RdCVFL protein comprises a re-encoded nucleotide sequence.

6. The nucleic acid according to claim 5, wherein the recoded nucleotide sequence lacks an start methionine codon.

7. The nucleic acid according to any one of claims 1 to 6, wherein the human IgK signal sequence is at the N-terminus of the RdCVFL protein.

8. The nucleic acid according to any one of claims 1 to 7, wherein a nucleotide sequence encoding a human IgK signaling sequence is operably linked to a nucleotide sequence encoding human RdCVFL.

9. The nucleic acid according to claim 1, wherein the RdCVFL protein and the human IgK signal sequence include an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 3 or SEQ ID NO:

15.

10. The nucleic acid according to claim 9, wherein the RdCVFL protein and the human IgK signal sequence include an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

3.

11. The nucleic acid according to claim 5, wherein the recoded nucleotide sequence has at least 40% of its codons recoded.

12. The nucleic acid according to claim 5, wherein the recoded nucleotide sequence differs from the corresponding native nucleotide sequence by at least 15% of its nucleotides.

13. The nucleic acid according to claim 5, wherein the recoded nucleotide sequence is less than 90% identical to the corresponding native nucleotide sequence.

14. The nucleic acid according to any one of claims 1 to 13, wherein the nucleotide sequence or recoded nucleotide sequence has one or more characteristics selected from the following: lack of a procaria inhibitory motif, lack of a consensus splice donor site, lack of a latent splice donor site, and having a GC content of 60-65%.

15. The nucleic acid according to any one of claims 1 to 14, wherein the promoter sequence is operably linked to a human IgK signal sequence and a nucleotide sequence encoding a human RdCVFL protein.

16. Promoter types include: phage-lambda (PL) promoter; SV40 initial promoter; herpes simplex virus (HSV) promoter; cytomegalovirus (CMV) promoter; hybrid promoters including CMV enhancer and chicken beta-actin promoter; tetracycline regulatory transactivator-responsive promoter (tet) system; long-terminal repeat (LTR) promoters such as MoMLV LTR, BIV LTR, or HIV LTR; Moloney's mouse sarcoma virus U3 region promoter; granzyme A promoter; regulatory sequences of the metallothionein gene; CD34 promoter; CD8 promoter; thymidine kinase (TK) promoter; B19 parvovirus promoter; PGK promoter; glucocorticoid promoter; heat shock protein (HSP) promoter; immunoglobulin promoter; MMTV promoter; Rouss sarcoma virus (RSV) promoter; lac promoter; CaMV The nucleic acid according to claim 15, selected from the 35S promoter; nopaline synthase promoter; MND promoter; and MNC promoter.

17. The nucleic acid according to claim 16, wherein the promoter is a CMV promoter.

18. The nucleic acid according to claim 17, wherein the CMV promoter comprises nucleotides 150 to 812 of SEQ ID NO:

2.

19. The nucleic acid according to any one of claims 1 to 18, wherein an intron sequence is operably linked to a sequence encoding an RdCVFL protein.

20. The nucleic acid according to claim 19, wherein the intron sequence is a beta-globin intron sequence.

21. The nucleic acid according to claim 20, wherein the intron sequence comprises nucleotides 820 to 1312 of SEQ ID NO:

2.

22. The nucleic acid according to any one of claims 1 to 21, comprising SEQ ID NO: 2, the nucleotide sequence of SEQ ID NO: 2 from 150 to 2044, or the nucleotide sequences of SEQ ID NO: 2 from 150 to 812, 820 to 1312, and 1340 to 2044.

23. A vector comprising the nucleic acid according to any one of claims 1 to 22.

24. The vector according to claim 23, which is a nonviral vector.

25. The vector according to claim 24, wherein the nonviral vector is selected from lipid nanoparticles (LNPs), highly branched poly(β-aminoesters) (HPAEs), single-chain cyclic polymers (SCKPs), poly(amidoamine) (PAMAM) dendrimers, and polyethyleneimines (PEIs).

26. The vector according to claim 23, which is a viral vector.

27. The vector according to claim 26, wherein the viral vector is selected from retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, herpesvirus vectors, hepatitis virus vectors, SV40 vectors, EBV vectors, and Newcastle disease virus vectors.

28. The vector according to claim 27, wherein the viral vector is an adeno-associated virus (AAV) vector.

29. The viral vector according to claim 28, wherein the AAV vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAVrh74.

30. The viral vector according to claim 29, wherein the AAV vector is AAV2.

31. The viral vector according to claim 29, wherein the AAV vector is AAV8.

32. The viral vector according to claim 26, which is not a bovine immunodeficiency virus vector.

33. An isolated cell comprising the nucleic acid described in any one of claims 1 to 22, wherein the cell is capable of secreting the RdCVFL protein.

34. A method for producing the RdCVFL protein, comprising culturing the cells described in claim 33 under conditions that enable the expression and secretion of the RdCVFL protein, and isolating the RdCVFL protein from the culture of the cells.

35. The method according to claim 34, further comprising purifying the RdCVFL protein from a cell culture.

36. A method for secreting RdCVFL protein from cells, comprising administering a nucleic acid according to any one of claims 1 to 22 or a vector according to any one of claims 23 to 32 to the cells under conditions that enable the expression and secretion of RdCVFL encoded by the nucleic acid or vector.

37. The isolated cells according to claim 33, wherein the cells are mammalian cells, or the method according to any one of claims 34 to 36.

38. The isolated cells according to claim 33, wherein the cells are human cells, or the method according to any one of claims 34 to 36.

39. The isolated cells or method according to claim 37, wherein the mammalian cells are eye cells.

40. The isolated cells or method according to claim 39, wherein the ophthalmic cells are selected from retinal pigment epithelial (RPE) cells, rod cells, cone cells, bipolar cells, horizontal cells, amacrine cells, ganglion cells, and ARPE-19 cells.

41. The isolated cells according to claim 33, wherein the cells are in vitro, or the method according to any one of claims 34 to 36.

42. The isolated cells according to claim 33, wherein the cells are in vivo, or the method according to any one of claims 34 to 36.

43. The isolated cells according to claim 33, wherein the cells are ex vivo, or the method according to any one of claims 34 to 36.

44. The isolated cells or method according to claim 37, wherein the mammalian cells are selected from 293 cells, CHO cells, PerC6 cells, Vero cells, BHK cells, HeLa cells, COS cells, MDCK cells, 3T3 cells, and WI38.

45. The isolated cells according to claim 33, or the method according to any one of claims 34 to 36, in which the cells are encapsulated.

46. A pharmaceutical preparation comprising (i) a pharmaceutically acceptable carrier, (ii) a nucleic acid according to any one of claims 1 to 22, a vector according to any one of claims 23 to 32, or (iii) a combination thereof.

47. A method for preserving rod cells and cone cells in the eye of a mammal, comprising administering to the eye of the mammal, in an amount effective for preserving rod cells and cone cells, a nucleic acid according to any one of claims 1 to 22, a vector according to any one of claims 23 to 32, a pharmaceutical composition according to claim 46, or a combination thereof.

48. The method according to claim 47, wherein the vector or nucleic acid is administered by subretinal injection.

49. AAV vector approximately 5 x 10 ８ ~Approx. 1×10 １１ The method according to claim 48, wherein a vector genome copy (GC) is administered by subretinal injection.

50. The method according to claim 47, wherein the vector or nucleic acid is administered by intravitreous injection, injection into the anterior chamber of the eye, subconjunctival injection, or sub-Tenon's capsule injection.

51. AAV vector approximately 5 x 10 ８ ~Approx. 5×10 １２ The method according to claim 50, wherein a vector genome copy (GC) is administered by intravitreal injection.

52. The method according to any one of claims 47 to 51, wherein the mammal is a human.

53. The method according to claim 52, wherein the mammal is suffering from an eye disease selected from the group consisting of retinal dystrophy, Stargardt disease, retinitis pigmentosa, age-related macular degeneration in dry state (AMD in dry state), geographic atrophy (advanced stage of AMD in dry state), age-related macular degeneration in wet state (AMD in wet state), glaucoma / ocular hypertension, diabetic retinopathy, Valday-Biedl syndrome, Bassen-Kohnzweig syndrome, Best's disease, choroidopathy, cerebral gynecomastia, congenital amaurosis, Refsun syndrome, Usher syndrome, thyroid-related eye disease, Graves' disease, disease related to retinal pigment epithelial cells, anterior segment disease, lens disease / cataract, eye cup syndrome, or uveitis.

54. The method according to any one of claims 47 to 53, wherein, prior to administration, the stored rod cells and pyramidal cells do not contain the nucleic acid described in any one of claims 1 to 22.

55. A method according to any one of claims 47 to 53, comprising administering a nucleic acid according to any one of claims 1 to 22 or a vector according to any one of claims 23 to 32 to the eye of a mammal, wherein the nucleic acid or the vector is administered by subretinal injection, and rod cells and cone cells are preserved at a location at least 1 mm from the site of subretinal injection.

56. The method according to claim 55, wherein rod cells are preserved at a location at least 2 mm from the site of subretinal injection.

57. A method for treating a disease, comprising administering to a mammal a nucleic acid according to any one of claims 1 to 22, a vector according to any one of claims 23 to 32, a pharmaceutical preparation according to claim 46, or a combination thereof, wherein the disease is a central nervous system (CNS) disease.

58. The method according to claim 57, wherein the CNS disorder is Alzheimer's disease, Huntington's disease, Parkinson's disease, or an olfactory disorder.

59. The method according to claim 57 or 58, wherein the administration includes intra-arachnoid injection.

60. AAV vector approximately 5 x 10 ８ ~Approx. 5×10 １４ The method according to claim 59, wherein a vector genome copy (GC) is administered by intraarachnoid injection.

61. The method according to claim 57 or 58, wherein the administration includes intravenous injection.

62. AAV vector approximately 5 x 10 ８ ~Approx. 1×10 １５ The method according to claim 61, wherein a vector genome copy (GC) is administered by intravenous injection.

63. The method according to any one of claims 57 to 62, wherein the mammal is a human.

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