Gene therapy for eye diseases

Abstract

Compositions and methods for the treatment of ocular diseases in a subject are provided. [Solution] In one aspect, an adeno-associated viral vector is provided, comprising a nucleic acid molecule comprising a sequence encoding CNGB3. In another aspect, an adeno-associated viral vector is provided, comprising a nucleic acid molecule comprising a sequence encoding CNGB3. In another aspect, an adeno-associated viral vector is provided, comprising a nucleic acid molecule comprising a sequence encoding REP-1. In a desirable embodiment, the subject is a human, cat, dog, sheep, or non-human primate.

Description

[Technical Field]

[0001] The present invention provides compositions and methods for the treatment of eye diseases in subjects. In one aspect, it provides an adeno-associated viral vector comprising a nucleic acid molecule containing a sequence encoding CNGA3. In another aspect, it provides an adeno-associated viral vector comprising a nucleic acid molecule containing a sequence encoding CNGB3. In yet another aspect, it provides an adeno-associated viral vector comprising a nucleic acid molecule containing a sequence encoding REP-1. In a preferred embodiment, the subject is a human, cat, dog, sheep, or non-human primate. [Background technology]

[0002] Reference to materials submitted in electronic format The applicant incorporates the sequence listing material submitted electronically as constituting part of this specification. This file is labeled “16-7660PCT_Seq_Listing_ST25.txt”.

[0003] Background of the Invention Choroideremia (CHM) is an X-linked hereditary retinal disease characterized by degeneration of photoreceptors, retinal pigment epithelium (RPE), and choroidal capillary tubules. Symptoms appear before the age of 10 or 20 with complaints of poor night vision (nyctalopia) and progressive loss of peripheral vision. The visual field narrows as the disease progresses. This peaks in some cases in the 30s with loss of central vision and blindness. More than 140 mutations in the CHM gene have been found to cause choroideremia. Mutations may result in the production of abnormally small, non-functional, and / or unstable Rab escort protein-1 (REP-1) protein, reduced protein function, or loss of REP-1 protein production. The absence of normal REP-1 disrupts the ability of Rab proteins to assist in intracellular trafficking. Impaired migration of proteins and organelles within cells leads to impaired cell function and premature death.

[0004] The CHM gene for colloideremia encodes Rab escort protein-1 (REP-1), a 653-amino acid protein involved in the regulation of membrane trafficking. Because the CHM locus is located on the X chromosome, colloideremia is typically diagnosed only in males. Female carriers of the disease are usually asymptomatic, but retinal examinations often reveal patchy degeneration of the retina and RPE, and female subjects may develop the disease depending on the degree of X-inactivation (lyonization) of their normal X chromosome. See also Non-Patent Document 1, incorporated herein by reference, and Non-Patent Document 2, incorporated herein by reference.

[0005] Color blindness is a heterogeneous group of autosomal recessive retinal diseases characterized by early onset of visual acuity loss, incomplete or complete color vision abnormalities, nystagmus, photoaversion, and loss of cone photoreceptor function. Approximately 80% of color blindness subjects exhibit mutations in the alpha or beta subunits (A3 and B3) of the cGMP regulatory cation channel cyclic nucleotide-sensitized channel (CNG) of cone photoreceptors. Cnga3-deficient mice, which are homologous to the human disease, reveal cone-specific loss of function leading to dysfunction and degeneration of affected cone photoreceptors.

[0006] Therefore, a composition useful for the expression of CNGA3 or CNGB3 in human subjects is needed. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Coussa, RG, Traboulsi, EI (2012) Choroideremia: a review of general findings and pathogenesis, Ophthalmic Genet 33(2):57-65 [Non-Patent Document 2] Vasireddy et al., AAV-mediated gene therapy for choroideremia:preclinical studies in personalized models.PLoS One.2013 May 7;8(5):e61396 [Overview of the project]

[0008] Outline of the invention Colloideremia (CHM) is an X-linked retinal degeneration that manifests before the age of 10 or 20, causing night blindness and loss of peripheral vision. The disease progresses throughout middle age, by which time most subjects become blind. CHM is a preferred target for gene augmentation therapy because the disease is due to the loss of function of Rab escort protein 1 (REP1), a protein essential for the health of retinal cells, encoded by the CHM gene. CHM cDNA can be packaged in recombinant adeno-associated virus (rAAV), which has an established track record in human gene therapy research. Furthermore, there are highly sensitive and quantitative assays to demonstrate the activity of REP1, including its ability to prenylate Rab proteins such as Rab27 and correct defects in Rab27 localization and trafficking due to the lack of prenylation in REP-1-deficient cells.

[0009] In one aspect, we provide a codon-optimized cDNA sequence encoding Rab escort protein-1 (REP-1). In one aspect, the codon-optimized cDNA sequence is a variant of SEQ ID NO: 3. In another aspect, the codon-optimized cDNA sequence is SEQ ID NO: 1. In yet another aspect, the cDNA sequence is codon-optimized for expression in humans.

[0010] In another embodiment, the expression cassette includes a codon-optimized nucleic acid sequence encoding REP-1. In one embodiment, the expression cassette includes the cDNA sequence of SEQ ID NO: 1. In yet another embodiment, the REP-1 coding sequence is located between the 5' and 3' AAV ITR sequences. In one embodiment, the vector genome includes all nucleic acid sequences between and including the 5' ITR and 3' ITR.

[0011] In another embodiment, an adeno-associated virus (AAV) vector is provided. The AAV vector comprises a nucleic acid sequence including an AAV capsid and an AAV inverted terminal repeat sequence, a nucleic acid sequence encoding human Rab escort protein-1 (REP-1), and an expression regulatory sequence that directs the expression of REP-1 in host cells. In one embodiment, the REP-1 sequence encodes the full-length REP-1 protein. In one embodiment, the REP-1 sequence is the protein sequence of Sequence ID No. 2.

[0012] In one aspect, a codon-optimized cDNA sequence encoding the cyclic nucleotide-sensitive channel alpha-3 (CNGA3) is provided. In one embodiment, the codon-optimized cDNA sequence is a variant sequence of SEQ ID NO: 13 or SEQ ID NO: 15. In another embodiment, the codon-optimized cDNA sequence is SEQ ID NO: 9 or SEQ ID NO: 11. In yet another embodiment, the cDNA sequence is codon-optimized for expression in humans.

[0013] In another aspect, the expression cassette includes a codon-optimized nucleic acid sequence encoding a cyclic nucleotide-sensitive channel alpha-3 (CNGA3). In one embodiment, the expression cassette includes the cDNA sequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15. In yet another embodiment, the CNGA3 coding sequence is located between the 5' and 3' AAV ITR sequences.

[0014] In another aspect, the expression cassette includes a codon-optimized nucleic acid sequence encoding a cyclic nucleotide-sensitive channel alpha-3 (CNGB3). In one embodiment, the expression cassette includes the cDNA sequence of SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 23. In yet another embodiment, the CNGB3 coding sequence is located between the 5' and 3' AAV ITR sequences.

[0015] In another embodiment, an adeno-associated virus (AAV) vector is provided. The AAV vector comprises a nucleic acid sequence including an AAV capsid and an AAV inverted terminal repeat sequence, a nucleic acid sequence encoding human CNGA3, and an expression regulatory sequence that directs the expression of CNGA3 in host cells. In one embodiment, the CNGA3 sequence encodes a full-length CNGA3 protein. In one embodiment, the CNGA3 sequence is the protein sequence of SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.

[0016] In another embodiment, an adeno-associated virus (AAV) vector is provided. The AAV vector comprises a nucleic acid sequence including an AAV capsid and an AAV inverted terminal repeat sequence, a nucleic acid sequence encoding human CNGB3, and an expression regulatory sequence that directs the expression of CNGB3 in host cells. In one embodiment, the CNGB3 sequence encodes a full-length CNGB3 protein. In one embodiment, the CNGB3 sequence is the protein sequence of Sequence ID No. 20.

[0017] In another aspect, we provide an adeno-associated virus (AAV) vector comprising an AAV8 capsid and an expression cassette, wherein the expression cassette comprises a nucleic acid sequence encoding REP-1, an inverted terminal repeat sequence, and an expression regulatory sequence that directs the expression of REP-1 in host cells.

[0018] In another aspect, we provide an adeno-associated virus (AAV) vector comprising an AAV8 capsid and an expression cassette, wherein the expression cassette comprises a nucleic acid sequence encoding CNGA3, an inverted terminal repeat sequence, and an expression regulatory sequence that directs the expression of CNGA3 in host cells.

[0019] In yet another aspect, we provide an adeno-associated virus (AAV) vector comprising an AAV8 capsid and an expression cassette, wherein the expression cassette comprises a nucleic acid sequence encoding CNGB3, an inverted terminal repeat sequence, and an expression regulatory sequence that directs the expression of CNGB3 in host cells.

[0020] In another aspect, we provide an adeno-associated virus (AAV) vector comprising an AAV2 capsid and an expression cassette, wherein the expression cassette comprises a nucleic acid sequence encoding REP-1, an inverted terminal repeat sequence, and an expression regulatory sequence that directs the expression of REP-1 in host cells.

[0021] In another aspect, we provide an adeno-associated virus (AAV) vector comprising an AAV2 capsid and an expression cassette, wherein the expression cassette comprises a nucleic acid sequence encoding CNGA3, an inverted terminal repeat sequence, and an expression regulatory sequence that directs the expression of CNGA3 in host cells.

[0022] In yet another aspect, we provide an adeno-associated virus (AAV) vector comprising an AAV2 capsid and an expression cassette, wherein the expression cassette comprises a nucleic acid sequence encoding CNGB3, an inverted terminal repeat sequence, and an expression regulatory sequence that directs the expression of CNGB3 in host cells.

[0023] In another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent, excipient and / or additive and at least one viral vector as described herein.

[0024] In further aspects, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent, excipient and / or additive, and a nucleic acid sequence, plasmid, vector or viral vector such as rAAV as specifically described herein.

[0025] In another aspect, a method for treating colloideremia is provided. In one embodiment, the method comprises administering a composition comprising an AAV vector encoding REP-1 as described herein to a subject in need.

[0026] In another aspect, a method for treating color blindness is provided. In one embodiment, the method involves administering a composition comprising an AAV vector encoding CNGA3 as described herein to a subject in need.

[0027] In another aspect, a method for treating color blindness is provided. In one embodiment, the method involves administering a composition comprising an AAV vector encoding CNGB3 as described herein to a subject in need.

[0028] In yet another aspect, plasmids for the construction of AAV vectors are provided. In one embodiment, the plasmid comprises a codon-optimized cDNA sequence encoding REP-1 as described herein. In another embodiment, the plasmid comprises a codon-optimized cDNA sequence encoding CNGA3 as described herein. In yet another embodiment, the plasmid comprises a codon-optimized cDNA sequence encoding CNGB3, which is a sequence sharing at least 70% identity with SEQ ID NO: 19 or SEQ ID NO: 21. In one embodiment, the plasmid is modular.

[0029] In another aspect, a method for producing rAAV viruses is provided. The method comprises culturing packaging cells possessing a plasmid described herein in the presence of sufficient viral sequences to enable packaging of a gene-expressing cassette viral genome within an infectious AAV envelope or capsid. In another aspect, recombinant AAV produced according to the method is provided.

[0030] Other aspects and advantages of the present invention will be readily apparent from the following detailed description of the invention. [Brief explanation of the drawing]

[0031] [Figure 1] Figures 1A and 1B show gels illustrating in vitro REP-1 protein expression in cultured 84-31HEK cells after transfection. The first lane of each gel shows the expression of the codon-optimized REP-1 described herein, expressed from plasmid p944. The second lane shows the expression of native REP-1 from plasmid p742. The third lane shows the endogenous expression of REP-1 by 84-31 cells that were not transfected with a plasmid. The last lane is blank. The gels demonstrate that the codon-optimized REP-1 sequence described herein produces higher levels of protein expression than the native REP-1 sequence, and that the level of expression from externally transfected plasmids is several times higher than that of endogenous REP-1 expression. [Figure 2] Figure 2 shows the alignment of the natural REP-1 coding sequence of sequence number 1 versus the codon-optimized REP-1 coding sequence of sequence number 3. [Figure 3] Figure 3 shows the alignment of the natural CNGA3 coding sequence of sequence number 13 versus the codon-optimized CNGA3 coding sequence of sequence number 9. [Figure 4] Figure 4 shows the alignment of a natural CNGB3 ORF (SEQ ID NO: 19) versus a modified CNGB3 ORF (SEQ ID NO: 21) versus a modified CNGB3 ORF with a modified end (SEQ ID NO: 23). Point mutations are highlighted. [Figure 5] Figure 5 is a plasmid map of p584 as described herein. The sequence of p584 is shown in Sequence ID No. 7. [Figure 6] Figure 6 is a plasmid map of AAV.hCHMco. ​​version 2a as described herein. The sequence of version 2a is shown in SEQ ID NO: 25. [Figure 7] Figure 7 is a plasmid map of AAV.hCHMco. ​​version 2b described herein. The sequence of version 2b is shown in SEQ ID NO: 26. [Figure 8]Figure 8 is a plasmid map of AAV.hCHMco. ​​version 3a as described herein. The sequence of version 3a is shown in SEQ ID NO: 27. [Figure 9] Figure 9 is a plasmid map of AAV.hCHMco. ​​version 3b described herein. The sequence of version 3b is shown in SEQ ID NO: 28. [Figure 10] Figure 10 is a plasmid map of AAV.hCHM. version 1 as described herein. The sequence of version 1 is shown in SEQ ID NO: 29. [Figure 11] Figure 11 is a graph showing the effect of lambda insert on AAV product impurities. From qPCR testing, all α-version (lambda-containing) vectors have a significantly reduced Kan+ signal. [Figure 12] Figure 12A is a Western blot showing the detection of ~75-80 kDa protein by human anti-REP-1 antibody in the eye tissue of CD-1 mice injected with AAV8.2b in 5 × 10⁹ (5E9) (high dose) vector genome copies. Animals injected with AAV8.2b in 5 × 10⁸ (low dose) showed a very weak protein band at ~75-80 kDa. Figure 12B is a Western blot analysis of the eye tissue of AAV8.3b injected CD1 mice (2 mice per group) detected using anti-REP-1 antibody, which revealed the presence of ~75-80 kDa protein in one eye injected with the low dose and in both eyes injected with the high dose of AAV8.3b. REP-1 expression was not detected in the eye tissue of mice that were not injected. [Modes for carrying out the invention]

[0032] Detailed description of the invention The methods and compositions described herein include compositions and methods for delivering optimized CHM encoding REP-1 to mammalian subjects for the treatment of eye diseases, primarily blindness disorders such as colloideremia. Furthermore, the methods and compositions described herein include compositions and methods for delivering optimized CNGA3 or CNGB3 to mammalian subjects for the treatment of eye diseases, primarily blindness disorders such as color blindness. In one embodiment, such a composition includes codon optimization of the REP-1, CNGA3, or CNGB3 coding sequence. These features are expected to improve the efficacy of the product and enhance safety as lower doses of reagent are used. This optimization of the transgene cassette may theoretically maximize the level of experimental protein production compared to the level that can be produced using endogenous sequences. However, compositions containing the natural REP1, CNGA3, and CNGB3 coding sequences, as shown in SEQ ID NO: 3, SEQ ID NO: 13, and SEQ ID NO: 19, are also included herein. Where embodiments are described with respect to any of REP-1, CNGA3, or CNGB3, it should be understood that similar embodiments are intended to be referenced elsewhere. That is the case.

[0033] The technical and scientific terms used herein have the same meaning as they would normally be understood by a person of ordinary skill in the art to which the invention pertains, and by reference to published books, which provide a general guide to many of the terms used herein. The definitions contained herein are given for clarity in the descriptions of components and compositions herein and are not intended to limit the claimed invention.

[0034] The colloideremia gene, CHM, encodes Rab escort protein-1 (REP-1), a 653-amino acid protein thought to be involved in membrane trafficking. As used herein, the terms "REP-1" and "CHM" are interchangeable when referring to the coding sequence. Because the CHM locus is located on the X chromosome, colloideremia is typically diagnosed only in males. Female carriers of the disease are usually asymptomatic, but retinal examinations often reveal patchy degeneration of the retina and RPE, and female subjects may be affected depending on the degree of X-inactivation (lyonization) of the normal X chromosome. See Coussa cited above. The native amino acid sequence encoding human REP-1 is reported in GenBank acceptance number P24386 and is reproduced herein by SEQ ID NO: 2. The native human nucleic acid sequence of CHM is reproduced herein by SEQ ID NO: 3 (acceptance number NM_000390.2).

[0035] Cyclic nucleotide-sensitive (CNG) ion channels are essential mediators underlying signaling in the retina and olfactory receptors. Genetic defects in CNGA3 and CNGB3, which encode two structurally related subunits of cone CNG channels, are known to cause color vision deficiency. CNGA3 is a 694-amino acid protein, and CNGB is an 809-amino acid protein.

[0036] Color blindness is a heterozygous group of congenital autosomal recessive retinal disorders that manifests as early-onset cone photoreceptor dysfunction, severely reduced visual acuity, impaired or complete color blindness, and photophobia. The native nucleic acid sequence encoding human CNGA3 is reported in GenBank acceptance number XM_011210554.1 and reproduced in Sequence ID No. 13. The native nucleic acid sequence encoding human CNGA3 is reported in GenBank acceptance number XM_011210554.1 and reproduced in Sequence ID No. 13. The native nucleic acid sequence for the human CNGA3 X1 variant, including an additional exon, is reported in GenBank acceptance number NM_001298.2 and reproduced in Sequence ID No. 15. The native nucleic acid sequence encoding human CNGB3 is reproduced in Sequence ID No. 19.

[0037] In some embodiments of the present invention, a subject has an “eye disease,” and the components, compositions, and methods of the present invention are designed for the treatment thereof. As used herein, the term “subject” means a mammal, including humans, veterinary or farm animals, livestock or pets, and animals commonly used for clinical research. In one embodiment, the subject of these methods and compositions is human. Further suitable subjects include, but are not limited to, mice, rats, dogs, cats, pigs, cattle, sheep, non-human primates, and others. As used herein, the term “subject” is interchangeable with “patient.”

[0038] As used herein, “eye disease” includes, but is not limited to, Stargardt disease (autosomal dominant or autosomal recessive), retinitis pigmentosa, and pattern dystrophy, as well as cone-rod dystrophies and retinal diseases. In one embodiment, the subject has color vision deficiency. In another embodiment, the subject This condition involves colloideremia or X-linked hereditary retinal degeneration. Clinical signs of such eye diseases include, but are not limited to, decreased peripheral vision, decreased central (reading) vision, decreased night vision, color vision loss, decreased visual acuity, decreased photoreceptor function, pigmentary changes, and ultimately blindness.

[0039] As used herein, the terms “treatment” or “treating” are defined to include administering to a subject one or more of the compounds or compositions described herein for the purpose of improving one or more symptoms of an eye disease. Thus, “treatment” may include one or more of the following in a given subject: mitigating the onset or progression of an eye disease, preventing the disease, reducing the severity of the symptoms of the disease or delaying their progression, including the progression of blindness, eliminating the symptoms of the disease, delaying the onset of the disease or monitoring the progression of the disease or the effectiveness of the treatment.

[0040] When used to describe nucleic acid sequences or proteins, the term “exogenous” means that the nucleic acid or protein does not naturally exist in the location where it is located within a chromosome or host cell. Exogenous nucleic acid sequences also refer to sequences that originate from the same host cell or subject and are inserted therein, but exist in a non-natural state, for example, at a different copy number or under the regulation of different regulatory factors.

[0041] When used to describe a nucleic acid sequence or protein, the term “heterologous” means that the nucleic acid or protein originates from a different organism or a different species of the same organism as the host cell or subject on which it is expressed. When used in reference to a protein or nucleic acid in a plasmid, expression cassette, or vector, the term “heterologous” means that the protein or nucleic acid exists together with another sequence or subsequence that the protein or nucleic acid in question would not naturally be found together with each other in the same relationship.

[0042] In the context of nucleic acid sequences, the terms “percent (%) identity,” “sequence identity,” “percent sequence identity,” or “percent identical” refer to bases in two sequences that are identical when aligned for correspondence. Percent identity is determined by comparing two sequences aligned under optimal conditions over the sequences to be compared. The length of the sequence identity comparison may be over the entire REP-1, CNGA3, or CNGB3 coding sequence or over a fragment of at least approximately 100–150 nucleotides, or as desired. However, identity between smaller fragments of, for example, at least approximately 9 nucleotides, typically at least approximately 20–24 nucleotides, at least approximately 28–32 nucleotides, or at least approximately 36 or more nucleotides may be desirable. Several sequence alignment programs are also available for nucleic acid sequences. Examples of such programs include “Clustal W,” “CAP Sequence Assembly,” “BLAST,” “MAP,” and “MEME,” which are available via web servers on the internet. Other sources of such programs are known to experts in the art. Alternatively, the Vector NTI utility may also be used. There are also several algorithms known in the art that can be used to measure nucleotide sequence identity, including those included in the above program. Another example is the Fasta program in GCG Version 6.1. TM Polynucleotide sequences can be compared using this method. Commonly available sequence analysis software, or more specifically, analytical tools provided by BLAST or public databases, may be used.

[0043] The term "isolated" means that a material has been taken out of its original environment (for example, its natural environment if it exists naturally). While naturally occurring polynucleotides or polypeptides are not isolated, the same polynucleotides or polypeptides isolated from some or all of the materials coexisting in nature are isolated, even if subsequently reintroduced into nature. Such polynucleotides may be part of a vector, and / or, such polynucleotides or polypeptides may be part of a composition, but are still isolated in that such vectors or compositions are not part of their natural environment.

[0044] "Engineered" means that the nucleic acid sequences encoding the REP-1, CNGA3, or CNGB3 proteins described herein are assembled and placed in any suitable genetic element, such as naked DNA, phage, transposon, cosmid, episome, etc., to transport and transfer the REP-1, CNGA3, or CNGB3 sequences into host cells, for example, to construct a non-viral delivery system (e.g., an RNA-based system, naked DNA, etc.), or to construct a viral vector in a packaging host cell, and / or to deliver it to a host cell in a subject. In one embodiment, the genetic element is a plasmid. The methods used for constructing such engineered constructs are known to experts in nucleic acid manipulation and include genetic engineering, recombination, and synthesis methods. For example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor. See Press, Cold Spring Harbor, NY (2012).

[0045] As used herein, the term “transgene” means an exogenous or modified protein-coding nucleic acid sequence that is under the control of a promoter or regulatory sequence in an expression cassette, rAAV genome, recombinant plasmid or production plasmid, vector or host cell as described herein. In some embodiments, the transgene is a human CHM(REP-1) sequence encoding a functional REP-1 protein. In some embodiments, the transgene is a codon-optimized nucleic acid CHM(REP-1) encoding the REP-1 amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the transgene is encoded by the sequence shown in SEQ ID NO: 1. In some embodiments, the REP-1 transgene is encoded by the sequence shown in SEQ ID NO: 5. SEQ ID NO: 5 includes a modified end which includes a restriction site for cloning into a plasmid, such as a production plasmid as described herein.

[0046] In some embodiments, the transgene is a human CNGA3 sequence encoding a functional CNGA3 protein. In some embodiments, the transgene is a codon-optimized CNGA3 encoding the sequence of SEQ ID NO: 10. In some embodiments, the transgene is encoded by the sequence shown in SEQ ID NO: 9. In one embodiment, the transgene includes a modified end, such as that shown in SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18, which includes a restriction site for cloning into a plasmid, such as the plasmid described herein. In some embodiments, the transgene is a codon-optimized CNGA3 encoding the sequence of SEQ ID NO: 12. In some embodiments, the transgene is encoded by the sequence shown in SEQ ID NO: 11. In some embodiments, the transgene is encoded by the native coding sequence of CNGA3 shown in SEQ ID NO: 13.

[0047] In some embodiments, the transgene is a human CNGB3 sequence encoding a functional CNGB3 protein. In some embodiments, the transgene is a codon-optimized CNGB3 encoding a sequence that shares at least 70% identity with SEQ ID NO: 19 or 21. In some embodiments, the transgene is encoded by the sequence shown in SEQ ID NO: 23. SEQ ID NO: 23 includes a modified end, which includes a restriction site for cloning into a plasmid such as the production plasmid described herein. Sequences 13 to 2448 provide ORFs for CNGB3. In some embodiments, the transgene is a codon-optimized CNGB3 encoding the sequence of Sequence ID No. 20. In some embodiments, the transgene is encoded by the sequence shown in Sequence ID No. 19. In some embodiments, the transgene is encoded by the sequence shown in Sequence ID No. 21. In some embodiments, the transgene includes modified ends for cloning into a plasmid, such as the plasmids described herein. Sequence ID No. 21 is a novel cDNA sequence in which a silent mutation has been introduced into the native coding sequence. Further modifications to the native sequences described herein are intended by the present invention.

[0048] In one embodiment, a nucleic acid sequence encoding REP-1, CNGA3, or CNGB3 further comprises a nucleic acid encoding a labeled polypeptide covalently bound thereto. The labeled polypeptide may be selected from known “epitope labels,” including but not limited to myc-labeled polypeptides, glutathione-S-transferase-labeled polypeptides, green fluorescent protein-labeled polypeptides, myc-pyruvate kinase-labeled polypeptides, His6-labeled polypeptides, influenza virus hemagglutinin-labeled polypeptides, flag-labeled polypeptides, and maltose-binding protein-labeled polypeptides.

[0049] As used herein, “vector” is a nucleic acid molecule that may insert an exogenous, heterologous, or engineered nucleic acid transgene into which it may then be introduced into a suitable host cell. A vector preferably has one or more origins of replication and one or more sites into which recombinant DNA may be inserted. Vectors often have a convenient means of selecting cells that have the vector from cells that do not, for example, they encode drug resistance genes. Common vectors include plasmids, viral genomes, and “artificial chromosomes” (mainly in yeast and bacteria). Some plasmids are described herein.

[0050] A “viral vector” is defined as a replication-deficient virus containing an exogenous or heterologous CHM(REP-1) or CNGA3 or CNGB3 nucleic acid transgene. In one embodiment, the expression cassette described herein may be engineered on a plasmid used for drug delivery or for the construction of a viral vector. A suitable viral vector is preferably replication-deficient and selected from those that target ophthalmic cells. Viral vectors include, but are not limited to, adenoviruses, herpesviruses, lentiviruses, retroviruses, and parvoviruses, and may include any virus suitable for gene therapy. However, for ease of understanding, adenoviruses are referred to herein as representative viral vectors.

[0051] A “replication-deficient virus” or “viral vector” refers to a synthetic or recombinant viral particle in which an expression cassette containing the gene in question is packaged within a viral capsid or envelope, and any viral genome sequence packaged within the viral capsid or envelope is replication-deficient; that is, they cannot produce progeny virions but retain the ability to infect target cells. In one embodiment, the genome of a viral vector does not contain genes encoding enzymes necessary for replication (the genome can be manipulated to be “gutless,” containing only the transgene of interest flanked by signals necessary for amplification and packaging of the artificial genome), but these genes may be supplied during production. Thus, since replication and infection by progeny virions cannot occur except in the presence of viral enzymes necessary for replication, it is considered safe for use in gene therapy.

[0052] In yet another embodiment, a recombinant AAV genome is constructed using an expression cassette containing any of those described herein.

[0053] As used herein, the term “host cell” may refer to a packaging cell line from which recombinant AAV is produced from a production plasmid. Alternatively, the term “host cell” may refer to any target cell in which the expression of the transgene is desired. Thus, “host cell” refers to a prokaryotic or eukaryotic cell containing exogenous or heterologous DNA introduced into the cell by any of the following means, e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion, high velosity DNA-coated pellet, viral infection, and protoplast fusion.

[0054] In some aspects of this specification, the term “host cells” refers to cultures of ophthalmic cells of various mammalian species for in vitro evaluation of the compositions described herein. In other aspects of this specification, the term “host cells” refers to cells used to produce and package viral vectors or recombinant viruses. In yet another aspect, the term “host cells” is intended to refer to ophthalmic cells of a subject being treated in vivo for an eye disease.

[0055] As used herein, the term “oculocellular cells” refers to any cells in the eye or associated with the function of the eye. The term may refer to any one of the following: photoreceptor cells, including rod photoreceptors, cone photoreceptors, and photosensitive ganglion cells; retinal pigment epithelial (RPE) cells; Müller cells; choroidal cells; bipolar cells; horizontal cells; and amacrine cells. In one embodiment, oculocellular cells are photoreceptor cells. In another embodiment, oculocellular cells are RPE cells.

[0056] "Plasmid" is generally referred to herein by a lowercase 'p' followed by an uppercase letter and / or a number, in accordance with standard nomenclature well known to those skilled in the art. Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known to those skilled in the art and readily available. Furthermore, skilled persons can readily construct any number of other plasmids suitable for use in the present invention. The properties, construction and use of such plasmids and other vectors in the present invention will be readily apparent to skilled persons from this disclosure.

[0057] As used herein, the terms “transcriptional regulatory sequence” or “expression regulatory sequence” refer to DNA sequences such as initiator sequences, enhancer sequences, and promoter sequences that induce, repress, or otherwise regulate the transcription of a protein-coding nucleic acid sequence to which they are operably linked.

[0058] As used herein, the terms “operably linked” or “operably associated” refer to both regulatory expression sequences that are contiguous with a nucleic acid sequence encoding REP-1 or CNGA3, and / or regulatory expression sequences that work in trans or away to regulate their transcription and expression.

[0059] As used herein, the terms “AAV” or “AAV serotype” refer to a large number of naturally occurring and available adenoviruses as well as artificially created AAVs. Among the AAVs isolated or engineered from humans or non-human primates (NHPs) and thoroughly characterized, human AAV2 was the first AAV developed as a gene transfer vector; it has been widely used for effective gene transfer experiments in various target tissues and animal models. Unless otherwise noted, the AAV capsids, ITRs and other selected AAV components described herein include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8, and AAVAnt The AAVs may be readily selected from among 80, any variant of any known or mentioned AAVs, or any AAV including but not limited to undiscovered AAVs or mixtures thereof of their variants or mixtures. See, for example, International Publication No. 2005 / 033321 incorporated herein by reference. In another embodiment, the AAV capsid is the AAV8bp capsid, which preferentially targets bipolar cells. See, for example, International Publication No. 2014 / 024282 incorporated herein by reference. In another embodiment, the AAV capsid is the AAV7m8 capsid, which exhibits preferential delivery to the outer retina. See, Dalkara et al., In Vivo-Directed Evolution of a New Adeno-Associated Virus for Therapeutic Outer Retinal Gene Delivery from the Vitreous, Sci Transl Med 5, 189ra76 (2013), incorporated herein by reference. In one embodiment, the AAV capsid is the AAV8 capsid. In another embodiment, the AAV capsid is the AAV9 capsid. In yet another embodiment, the AAV capsid is the AAV5 capsid. In yet another embodiment, the AAV capsid is the AAV2 capsid.

[0060] Where used herein with respect to AAV, the term "variant" means an AAV sequence derived from a known AAV sequence, including those that share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or higher sequence identity across the entire amino acid or nucleic acid sequence. In other embodiments, an AAV capsid includes a variant that may contain up to about 10% mutations from any described or known AAV capsid sequence. That is, an AAV capsid shares about 90% to about 99.9% identity, about 95% to about 99% identity, or about 97% to about 98% identity with an AAV capsid presented herein and / or known in the art. In one embodiment, an AAV capsid shares at least 95% identity with an AAV capsid. When determining the percentage identity of an AAV capsid, comparisons may be made with any of several variable proteins (e.g., vp1, vp2, or vp3). In one embodiment, the AAV capsid shares at least 95% identity with AAV8 vp3. In another embodiment, a self-complementary AAV is used.

[0061] ITRs or other AAV components may be readily isolated or manipulated from AAV using methods available to those skilled in the art. Such AAV may be isolated, manipulated, or obtained from academic, commercial, or public sources (e.g., American Type Culture Collection, Manassas, VA). Alternatively, AAV sequences may be manipulated synthetically or by other appropriate means by reference to publicly available sequences, such as those available in the literature or in databases such as GenBank or PubMed. Manipulating AAV viruses by conventional molecular biological methods may allow for the optimization of these particles for purposes such as cell-specific delivery of nucleic acid sequences, minimizing immunogenicity, adjusting stability and particle lifetime, effective degradation, and precise delivery to the nucleus.

[0062] As used herein, “artificial AAV” means, but is not limited to, an AAV having a capsid protein that does not exist naturally. Such artificial capsids may be constructed by appropriate means using a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) in combination with heterologous sequences that may be obtained from different selected AAVs, non-continuous portions of the same AAV, or non-AAV viral sources or non-viral sources. Artificial AAVs may be pseudotyped AAVs, chimeric AAV capsids, recombinant AAV capsids, or “humanized” AAV capsids. In some cases, but not limited to, pseudotype vectors in which the capsid of one type of AAV is replaced with a different type of capsid protein are useful in the present invention. In one embodiment, AAV2 / 5 and AAV2 / 8 are typical pseudotype vectors.

[0063] "Self-complementary AAV" refers to a plasmid or vector having an expression cassette designed so that the coding region of the recombinant AAV nucleic acid sequence forms an intramolecular double-stranded DNA template. Upon infection, rather than waiting for cell-mediated synthesis of the second strand, the two complementary halves of scAAV will associate to form a single double-stranded DNA (dsDNA) unit ready for immediate replication and transcription. See, for example, DM McCarty et al., “Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis”, Gene Therapy, (August 2001), Vol 8, Number 16, pages 1248-1254. Self-complementary AAVs are described, for example, in U.S. Patents 6,596,535; 7,125,717 and 7,456,683, the entirety of which is incorporated herein by reference.

[0064] In the methods, “administration” means delivering the composition to target-selected cells characterized by an eye disease. In one embodiment, the method includes delivering the composition to RPE, photoreceptor cells, or other ophthalmic cells by subretinal injection. In another embodiment, intravitreal injection into ophthalmic cells is used. In yet another embodiment, injection into ophthalmic cells via the palpebral vein may be used. Still further methods of administration may be selected by a person skilled in the art given this disclosure. “Administration” or “route of administration” is the delivery of the compositions described herein to a subject, with or without pharmaceutical carriers or excipients. Routes of administration may be combined if desired. In some embodiments, administration is repeated periodically. The pharmaceutical compositions described herein are designed for delivery to a subject in need by any suitable route or a combination of various routes. Direct delivery to the eye (optionally via intraocular delivery, subretinal injection, intraretinal injection, intravitreous, or local delivery) or delivery via systemic routes, such as intra-arterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral administration routes. Nucleic acid molecules and / or vectors described herein may be delivered in a single composition or in multiple compositions. Optionally, two or more AAVs or multiple viruses may be delivered [see, for example, International Publication No. 2011 / 126808 and International Publication No. 2013 / 049493]. In another embodiment, the multiple viruses may include different replication-deficient viruses (e.g., AAV and adenovirus) individually or in combination with proteins.

[0065] Some of the compositions described herein are isolated or synthetically or recombinantly manipulated nucleic acid sequences that give novel codon-optimized sequences encoding REP-1 or CNGA3 or CNGB3. In one embodiment, an isolated or manipulated codon-optimized nucleic acid sequence encoding human REP-1 is provided. In one embodiment, the codon-optimized sequence is Sequence ID No. 1. In another embodiment, the codon-optimized sequence includes N-terminal and C-terminal restriction sites for cloning. In one embodiment, as disclosed in Sequence ID No. 5, the REP-1 coding sequence includes an N-terminal NotI restriction site and a C-terminal BamHI restriction site in addition to the Kozak common sequence. In several further embodiments, the codon-optimized sequence includes one or more additional restriction sites to allow the addition of markers such as epitope labeling. When placed alongside a native nucleic acid sequence, the codon-optimized REP-1 may have at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% percent identity, including any integer between the ranges. In one embodiment, Codon-optimized REP-1 exhibits at least 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% percent identity with the natural sequence. In one embodiment, when placed side-by-side with the natural nucleic acid sequence number 3, codon-optimized REP-1 (sequence number 1) was found to have only 74% percent sequence identity (see Figure 2).

[0066] In another embodiment, isolated or manipulated codon-optimized nucleic acid sequences encoding human CNGA3 are provided. In one embodiment, the codon-optimized sequence is Sequence ID No. 9. In one embodiment, the codon-optimized sequence is a CNGA3 variant shown in Sequence ID No. 11. In another embodiment, the codon-optimized sequence includes N-terminal and C-terminal restriction sites for cloning. In one embodiment, the CNGA3 coding sequence includes an N-terminal NotI restriction site and a C-terminal BgIII restriction site in addition to the Kozak common sequence. Examples of CNGA3 sequences including such modifications can be found in Sequence ID No. 16, Sequence ID No. 17 and Sequence ID No. 18. In several further embodiments, the codon-optimized sequence includes one or more additional restriction sites to allow the addition of markers such as epitope labeling. When placed alongside the native nucleic acid sequence, the codon-optimized CNGA3 may have at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% percent identity, including any integer between the ranges. In one embodiment, codon-optimized CNGA3 has at least 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% percent identity with the natural sequence. In one embodiment, when placed side by side with the natural nucleic acid sequence number 13, codon-optimized CNGA3 (sequence number 9) was found to have only 80% percent sequence identity (see Figure 3).

[0067] In another embodiment, isolated or manipulated codon-optimized nucleic acid sequences encoding human CNGB3 are provided. In one embodiment, the codon-optimized sequence is a sequence that shares at least 70% identity with SEQ ID NO: 19 or SEQ ID NO: 21. In another embodiment, the codon-optimized sequence includes N-terminal and C-terminal restriction sites for cloning, as shown, for example, SEQ ID NO: 23. In several further embodiments, the codon-optimized sequence includes one or more additional restriction sites to allow the addition of markers, such as epitope labeling. When placed alongside a native nucleic acid sequence (as shown in SEQ ID NO: 19), the codon-optimized CNGB3 may have at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% percent identity, including any integer between the ranges. In one embodiment, the codon-optimized CNGB3 has at least 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% percent identity with the native sequence.

[0068] In one embodiment, an optimized nucleic acid sequence encoding the REP-1 or CNGA3 construct described herein is manipulated and introduced into a suitable genetic element, such as naked DNA, a phage, a transposon, a cosmid, an RNA molecule (e.g., mRNA), or an episome, which then transfer the REP-1 or CNGA3 sequence it contains to a host cell for, for example, to produce a viral vector in a host cell containing DNA or RNA, and / or to deliver it to a host cell in a subject. In one embodiment, the genetic element is a plasmid.

[0069] The selected genetic elements may be delivered by any appropriate method, including transfection, electroporation, liposome delivery, membrane fusion, rapid DNA-coated pellets, viral infection, and protoplast fusion. The methods used to construct such constructs are known to experts in nucleic acid manipulation and include genetic engineering, recombination, and synthesis. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0070] A variety of expression cassettes are provided that use SEQ ID NO: 1 or 5 for the expression of the REP-1 protein. In one embodiment, an example of a plasmid containing such an expression cassette is shown in SEQ ID NO: 25. In one embodiment, an example of a plasmid containing such an expression cassette is shown in SEQ ID NO: 26. In one embodiment, an example of a plasmid containing such an expression cassette is shown in SEQ ID NO: 27. In one embodiment, an example of a plasmid containing such an expression cassette is shown in SEQ ID NO: 28. As used herein, “vector genome” is a nucleic acid sequence that includes the ITRs themselves and is packaged between the 5' and 3' ITRs. In some embodiments, the term “vector genome” is used interchangeably with “expression cassette”. Thus, in one embodiment, the vector genome includes the 5' ITR, CMV enhancer, chicken beta-actin promoter, CBA exon 1 and intron, Kozak sequence, codon-optimized CHM, bGH poly-A, and the 3' ITR. In one embodiment, the vector genome includes nucleotides (nt) 1-4233 of SEQ ID NO: 25. In another embodiment, the vector genome includes nucleotides 1-4233 of SEQ ID NO: 26. In another embodiment, the vector genome contains nucleotides 1-4233 of SEQ ID NO: 27. In another embodiment, the vector genome contains nucleotides 1-4233 of SEQ ID NO: 28.

[0071] In another embodiment, a variety of expression cassettes are provided using SEQ ID NOs: 9, 11, or 13 for the expression of the CNGA3 protein. In another embodiment, a variety of expression cassettes are provided using SEQ ID NOs: 19, 21, or 23 for the expression of the CNGAB protein. As used herein, “expression cassette” refers to a nucleic acid molecule comprising a coding sequence, promoter, and possibly other regulatory sequences for an optimized REP-1 or CNGA3 or CNGB3 protein, which may be manipulated and introduced into a genetic element or plasmid and / or packaged in the capsid of a viral vector (e.g., a viral particle). In one embodiment, the expression cassette comprises a codon-optimized nucleic acid sequence encoding REP-1. In one embodiment, the cassette provides codon-optimized REP-1 operably associated with an expression regulatory sequence that directs the expression of the codon-optimized nucleic acid sequence encoding REP-1 in a host cell.

[0072] In another embodiment, the expression cassette comprises a codon-optimized nucleic acid sequence encoding CNGA3. In one embodiment, the cassette provides codon-optimized CNGA3 operably associated with an expression regulatory sequence that directs the expression of the codon-optimized nucleic acid sequence encoding CNGA3 in a host cell.

[0073] In another embodiment, the expression cassette comprises a codon-optimized nucleic acid sequence encoding CNGB3. In one embodiment, the cassette provides codon-optimized CNGB3 operably associated with an expression regulatory sequence that directs the expression of the codon-optimized nucleic acid sequence encoding CNGB3 in a host cell.

[0074] In another embodiment, an expression cassette for use in an AAV vector is provided. In one embodiment, the AAV expression cassette includes at least one AAV inverted end repeat (ITR) sequence. In another embodiment, the expression cassette includes a 5' ITR sequence and a 3' ITR sequence. In one embodiment, the 5' and 3' ITRs flank to a codon-optimized nucleic acid sequence encoding REP-1 or CNGA3 or CNGB3, together with an additional sequence that optionally directs the expression of the codon-optimized nucleic acid sequence encoding REP-1 or CNGA3 or CNGB3 in the host cell. Thus, as described herein, the AAV expression cassette describes the expression cassette described above, flanked by a 5' AAV inverted end repeat (ITR) at its 5' end and a 3' AAVITR at its 3' end. Thus, this rAAV genome includes the minimum sequences necessary to package the expression cassette within an AAV virus particle, namely the AAV 5' and 3' ITRs. The AAVITRs may be obtained from any AAV ITR sequence as described herein. These ITRs may be of the same AAV origin as the capsid used in the resulting recombinant AAV, or (for the creation of AAV pseudotypes) of a different AAV origin. In one embodiment, an ITR sequence from AAV2 or a deletion version thereof (ΔITR) is used for convenience and to expedite regulatory approval. However, ITRs from other AAV sources may be selected. Each rAAV genome may then be introduced into a production plasmid. In one embodiment, the production plasmid is one described herein or in International Publication No. 2012 / 158757, which is incorporated herein by reference. Various plasmids are known in the art for use in the construction of rAAV vectors and are useful herein. The production plasmid is cultured in host cells expressing AAV cap and / or rep proteins. In the host cells, each rAAV genome is rescued and packaged in a capsid protein or envelope protein to form an infectious viral particle.

[0075] One type of production plasmid is shown in Sequence ID No. 7, which is called p584. This plasmid is used in the examples for the construction of the rAAV-REP-1 vector. Such a plasmid contains a 5'AAVITR sequence; a selected promoter; a poly-A sequence; and a 3'ITR; it also contains a lambda-like stuffer sequence. In one embodiment, the non-coding lambda-stuffer region is included in the vector backbone. The nucleic acid sequence encoding REP-1, CNGA3, or CNGB3 is inserted between the selected promoter and the poly-A sequence or in place of a similar plasmid. An example of p584 containing the REP-1 coding sequence can be found in Sequence ID No. 8. In another embodiment, the production plasmid is modified for optimized vector plasmid production efficiency. Such modifications include the addition of other neutral sequences to regulate the level of supercoil in the vector plasmid or the deletion of part or all of the lambda-stuffer sequence. Such modifications are included herein. In other embodiments, a terminator and other sequences are included in the plasmid.

[0076] In a further embodiment, recombinant adeno-associated virus (AAV) vectors are provided for the delivery of REP-1, CNGA3, and CNGB3 constructs and optimized sequences as described herein. The adeno-associated virus (AAV) vector is an AAV DNase-resistant particle having an AAV protein capsid, in which the nucleic acid sequence is packaged within the capsid for delivery to target cells. The AAV capsid consists of 60 capsid (cap) protein subunits, VP1, VP2, and VP3, which are arranged in icosahedral symmetry in a ratio of approximately 1:1:10 to 1:1:20 depending on the selected AAV. AAVs may be selected as sources for the capsid of the AAV viral vector as defined above. See, for example, U.S. Published Patent Application No. 2007-0036760-A1; U.S. Published Patent Application No. 2009-0197338-A1; and European Patent No. 1310571. International Publication No. 2003 / 04239 See also Brochure No. 7 (AAV7 and other monkey AAVs), U.S. Patent No. 7,790,449 and U.S. Patent No. 7,282,199 (AAV8), International Publication No. 2005 / 033321 and U.S. Patent No. 7,906,111 (AAV9), and International Publication No. 2006 / 110,689 and International Publication No. 2003 / 042,397 (rh.10). These documents also describe other AAVs that may be selected for the creation of AAVs, and their contents become part of this specification by reference. In some embodiments, AAVs for use in viral vectors The cap may be produced by mutagenesis (i.e., insertion, deletion, or substitution) of one of the AAV capsids or its coding nucleic acids mentioned above. In some embodiments, the AAV capsid is a chimera containing domains from two, three, four, or more of the AAV capsid proteins mentioned above. In some embodiments, the AAV capsid is a mosaic of Vp1, Vp2, and Vp3 monomers from two or three AAVs or recombinant AAVs. In some embodiments, the rAAV composition contains more than one of the Caps mentioned above.

[0077] In another embodiment, the AAV capsid may include variants that may contain up to about 10% mutations from any of the described or known AAV capsid sequences. That is, the AAV capsid may share about 90%–99.9% identity, about 95%–99% identity, or about 97%–98% identity with the AAV capsids presented herein and / or known in the art. In one embodiment, the AAV capsid shares at least 95% identity with the AAV capsid. When determining the percentage identity of the AAV capsid, comparisons may also be made with any of the various proteins (e.g., vp1, vp2, or vp3). In one embodiment, the AAV capsid shares at least 95% identity with AAV8 vp3. In another embodiment, self-complementary AAVs are used. In one embodiment, it is desirable to use an AAV capsid that exhibits affinity (tropism) to a desired target cell, such as a photoreceptor, RPE, or other ophthalmic cell. In one embodiment, the AAV capsid is a tyrosine capsid-mutant in which some surface-exposed tyrosine residues are replaced with phenylalanine (F). Such AAV mutants include, for example, Mowat et al., Tyrosine capsid-mutant AAV vectors for gene delivery to the canine retina from a subretinal or intravitreal. approach,Gene Therapy 21,96-105(January This is described in (2014) and is incorporated herein by reference.

[0078] For packaging an expression cassette or rAAV genome or production plasmid into a virion, ITRs are the only AAV components required in cis within the same construct as the transgene. In one embodiment, coding sequences for replication (rep) and / or capsid (cap) are removed from the AAV genome and supplied trans or by a packaging cell line for the construction of the AAV vector. As described above, for example, pseudotyped AAV may contain ITRs from a different source than the source of the AAV capsid. Furthermore or alternatively, chimeric AAV capsids may be used. Yet another AAV component may be selected. Sources of such AAV sequences are described herein and may be isolated, manipulated or obtained from academic, commercial or public sources (e.g., American Type Culture Collection, Manassas, VA). Alternatively, AAV sequences may be obtained synthetically or by other suitable means by reference to publicly available sequences, such as those available in the literature or in databases such as GenBank®, PubMed®, etc.

[0079] Methods for producing and isolating AAV virus vectors suitable for delivery to subjects are in the relevant technical field. This is known in the following: For example, see U.S. Patent No. 7,790,449; U.S. Patent No. 7,282,199; International Publication No. 2003 / 042397; International Publication No. 2005 / 033321; International Publication No. 2006 / 110689; and U.S. Patent No. 7,588,772,B2. In one system, a producer cell line is transiently transfected with a construct encoding a transgene flanked by ITRs and a construct encoding rep and cap. In a second system, a packaging cell line that stably supplies rep and cap is transiently transfected with a construct encoding a transgene flanked by ITRs. In each of these systems, AAV virions are produced in response to infection with a helper adenovirus or herpesvirus that requires the isolation of rAAVs from the contaminating virus. More recently, systems have been developed that do not require helper virus infection for AAV recovery—the necessary helper functions (i.e., adenoviruses E1, E2a, VA, and E4 or herpesviruses UL5, UL8, UL52, and UL29, as well as herpesvirus polymerases) are also supplied transfectively by the system. In these newer systems, the helper functions may be supplied by transient transfection of cells using constructs encoding the necessary helper functions, or cells can be manipulated to stably contain genes encoding the helper functions, and the expression of those genes can be regulated at the transcriptional or post-transcriptional level.

[0080] In yet another system, the transgene and rep / cap gene flanked by ITRs are introduced into insect cells by infection using a baclovirus-based vector. For a review of these production systems, see, for example, Zhang et al., 2009, "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, the entire contents of which are incorporated herein by reference. Methods for the preparation and use of these and other AAV production systems are also described in the following U.S. patents, the entirety of which is incorporated herein by reference: 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439,065. Generally, for example, Grieger and Samulski, 2005, “Adeno-associated virus as a gene therapy”. Please refer to “vector: Vector development, production and clinical applications,” Adv. Biochem.Engin / Biotechnol.99:119-145; Buning et al., 2008, “Recent developments in adeno-associated virus vector technology,” J. Gene Med.10:717-733 and the references cited below, the entire contents of each of these are incorporated herein by reference.

[0081] The methods used to construct embodiments of the present invention are known to experts in nucleic acid manipulation and include genetic manipulation, recombination, and synthesis methods. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Similarly, methods for preparing rAAV virions are well known, and the selection of an appropriate method is not a limitation of the present invention. See, for example, K. Fisher et al., (1993) J. Virol., 70:520-532 and U.S. Patent No. 5,478,745.

[0082] An rAAV vector comprises an AAV capsid and an AAV expression cassette containing a sequence encoding REP-1, CNGA3, or CNGB3 as described above. In some embodiments, the rAAV expression cassette includes an AAV inverted terminal repeat sequence, a codon-optimized nucleic acid sequence encoding REP-1, CNGA3, or CNGB3, and an expression regulatory sequence that directs the expression of the encoded protein in the host cell. In other embodiments, the rAAV expression cassette further comprises one or more introns, Kozak sequences, poly(A), and post-transcriptional regulatory elements. Such rAAV vectors for use in pharmaceutical compositions for delivery to the eye may use a capsid derived from one of the many known AAVs defined above.

[0083] Other conventional components of expression cassettes and vectors include other components that can be optimized for specific species using methods known in the art, including codon optimization as described herein. Components of cassettes, vectors, plasmids and viruses or other compositions described herein include promoter sequences as part of the expression regulatory sequences. In another embodiment, the promoter is cell-specific. The term "cell-specific" means that a particular promoter selected for a recombinant vector may direct the expression of an optimized REP-1 or CNGA3 or CNGB3 transgene in a particular ophthalmic cell type. In one embodiment, the promoter is specific with respect to the expression of the transgene in photoreceptor cells. In another embodiment, the promoter is specific with respect to expression in rods and cones. In another embodiment, the promoter is specific with respect to expression in rods. In another embodiment, the promoter is specific with respect to expression in cones. In one embodiment, the photoreceptor-specific promoter is a human rhodopsin kinase promoter. The rhodopsin kinase promoter has been shown to be active in both rods and cones. See, for example, Sun et al., Gene Therapy with a Promoter Targeting Both Rods and Cones Rescues Retinal Degeneration Caused by AIPL1 Mutations, Gene Ther. 2010 January;17(1):117-131, which is incorporated in its entirety herein by reference. In one embodiment, the promoter is modified to add one or more restriction sites to facilitate cloning.

[0084] In another embodiment, the promoter is a human rhodopsin promoter. In one embodiment, the promoter is modified to include a restriction at its end for cloning. For example, the entire sequence of Nathans and Hogness, Isolation and nucleotide sequence of the See gene encoding human rhodopsin, PNAS, 81:4851-5 (August 1984). In another embodiment, the promoter is a part or fragment of the human rhodopsin promoter. In another embodiment, the promoter is a variant of the human rhodopsin promoter.

[0085] Other representative promoters include the human G protein-coupled receptor protein kinase 1 (GRK1) promoter (Genbank acceptance number AY327580). In another embodiment, the promoter is a 292-nucleotide fragment (positions 1793-2087) of the GRK1 promoter (see Beltran et al., Gene Therapy 2010 17:1162-74, the whole of which is incorporated herein by reference). In another preferred embodiment, the promoter is the human photoreceptor-retinoid-binding protein proximal (IRBP) promoter. In one embodiment, the promoter is a 235-nucleotide fragment of the hIRBP promoter. In one embodiment, the promoter is the RPGR proximal promoter (see Shu et al., IOVS, May 2010, the whole of which is incorporated herein by reference). Other promoters include the rod opsin promoter, red-green opsin promoter, blue opsin promoter, cGMP-β-phosphodiesterase promoter (Qgueta et al., IOVS, Invest Ophthalmol Vis Sci. 2000 Dec;41(13):4059-63), mouse opsin promoter (Beltran et al 2010 cited above), rhodopsin promoter (Mussolino et al., Gene Ther, July 2011, 18(7):637-45); cone transjucin alpha-subunit (Morrissey et al., BMC Dev, Biol, Jan 2011, 11:3); beta-phosphodiesterase (PDE) promoter; retinitis pigmentosa (RP1) promoter (Nicord et al., J. Gene Med, Dec 2007, 9(12):1015-23); NXNL2 / NXNL1 promoter (Lambard et al., PloS This includes, but is not limited to, the RPE65 promoter (One, Oct. 2010, 5(10):el3025), the retinal degeneration slow / peripherin 2 (Rds / perph2) promoter (Cai et al., Exp Eye Res. 2010 Ayg; 91(2):186-94), and the VMD2 promoter (Kachi et al., Human Gene Therapy, 2009 (20:31-9)). Each of these documents is incorporated herein by reference in its entirety. In another embodiment, the promoter is selected from the human EF1α promoter, rhodopsin promoter, rhodopsin kinase, photoreceptor-binding protein (IRBP), cone opsin promoters (red-green, blue), cone opsin upstream sequences including red-green cone locus regulatory regions, cone transduction and transcription factor promoters (neural retina leucine zipper (Nrl) and photoreceptor-specific nuclear receptor Nr2e3, bZIP).

[0086] In another embodiment, the promoter is either ubiquitous or constitutive. An example of a suitable promoter is the hybrid chicken β-actin (CBA) promoter with a cytomegalovirus (CMV) enhancer element. In another embodiment, the promoter is the CB7 promoter. Other suitable promoters include the human β-actin promoter, the human elongation factor-1α promoter, the cytomegalovirus (CMV) promoter, the monkey virus 40 promoter, and the herpes simplex virus thymidine kinase promoter. See, for example, Damdindorj et al., (August 2014) A Comparative Analysis of Constitutive Promoters Located in Adeno-Associated Viral Vecors. PloS ONE 9(8):e106472. Still other suitable promoters include viral promoters, constitutive promoters, and regulatable promoters [see, for example, International Publication No. 2011 / 126808 and International Publication No. 2013 / 04943]. Alternatively, promoters responsive to physiological cues may be used in the expression cassettes, rAAV genomes, vectors, plasmids, and viruses described herein. In one embodiment, due to the size limitations of the AAV vector, the promoter is small in size, less than 1000 base pairs (bp). In another embodiment, the promoter is smaller than 400 bp. Other promoters may be selected by experts in the art. In one embodiment, a ubiquitous promoter is introduced into the REP-1 construct. In another embodiment, a photoreceptor-specific promoter is introduced into the CNGA3 construct. In one embodiment, the REP-1 construct includes a CBA promoter together with a CMV enhancer element.

[0087] In another embodiment, the promoter is an inducible promoter. The inducible promoter may be selected from known promoters, including the rapamycin / rapalog promoter, the ecdysone promoter, the estrogen-reactive promoter, and the tetracycline-reactive promoter or a heterodimer repressor switch. Yes. The following are incorporated herein by reference in their entirety: Sochor et al., An Autogenously Regulated Expression System for Gene Therapeutic Ocular Applications. Scientific Reports. 2015 Nov 24;5:17105 and Daber R, Lewis M., A novel molecular switch. J Mol Biol 2009 Aug 28;391(4):661-70,Epub Please refer to June 21, 2009.

[0088] In another embodiment, the cassettes, vectors, plasmids, and viral constructs described herein include effective RNA processing signals such as other suitable transcription start, termination, enhancer sequences, splicing, and polyadenylation (poly-A) signals, TATA sequences, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (i.e., Kozak common sequences), introns, sequences that enhance protein stability, and, if desired, sequences that enhance the secretion of the encoded product. Expression cassettes or vectors may contain none of the elements described herein, or one or more of them. Examples of suitable poly-A sequences include, for example, SV40, bovine growth hormone (bGH), and TK poly-A. Examples of suitable enhancers include, among others, CMV enhancer, RSV enhancer, alpha-fetoprotein enhancer, TTR minimal promoter / enhancer, and LSP (TH-binding globulin promoter / alpha-microglobulin / bikunin enhancer). In one embodiment, a Kozak sequence is included upstream of the transgene coding sequence to enhance translation from the correct start codon. In another embodiment, CBA exon 1 and an intron are included in the expression cassette. In one embodiment, the transgene is placed under the regulation of a hybrid chicken β-actin (CBA) promoter. This promoter consists of a cytomegalovirus (CMV) early enhancer, a proximal chicken β-actin promoter, and CBA exon 1 flanked by an intron 1 sequence.

[0089] In one embodiment, the expression cassette comprises a 5'ITR, a CBA promoter, a CMV enhancer, CBA exon 1 and introns, a Kozak sequence, a human codon-optimized CHM sequence (SEQ ID NO: 1), a bGH polyA sequence, and a 3'ITR.

[0090] In another aspect, these nucleic acid sequences, vectors, expression cassettes, and rAAV virus vectors are useful in pharmaceutical compositions, which include pharmaceutically acceptable carriers, buffers, diluents, and / or additives. Such pharmaceutical compositions are used to express optimized REP-1, CNGA3, or CNGB3 in ophthalmic cells via delivery by such recombinantly engineered AAVs or artificial AAVs.

[0091] For the preparation of these pharmaceutical compositions comprising nucleic acid sequences, vectors, expression cassettes, and rAAV viral vectors, the sequences or vectors or viral vectors are preferably evaluated for contamination by conventional methods and then prepared into pharmaceutical compositions suitable for ocular administration. Such preparations include a pharmaceutically and / or physiologically acceptable vehicle or carrier, in particular a buffered saline or other buffer to maintain pH at an appropriate physiological level, such as HEPES, which is suitable for ocular administration, and optionally other medicinal products. This includes the use of agents, pharmaceutical agents, stabilizers, buffers, carriers, additives, diluents, etc. For injection, the carrier will typically be a liquid. Typical physiologically acceptable carriers include pyrogenically free sterile water and pyrogenically free sterile phosphate-buffered saline. A variety of such known carriers are provided in U.S. Patent Publication No. 7,629,322, which is incorporated herein by reference. In one embodiment, the carrier is an isotonic sodium chloride solution. In another embodiment, the carrier is an equilibrium salt solution. In one embodiment, the carrier includes tween viruses. If it needs to be stored for a long period, it may be frozen in the presence of glycerol or Tween20.

[0092] In one representative specific embodiment, the carrier or excipient composition contains 180 mM NaCl, 10 mM NaPi, pH 7.3, along with 0.0001%–0.01% Pluronic F68 (PF68). The exact composition of the buffer's saline component is in the range of 160 mM–180 mM NaCl. Optionally, a different pH buffer (possibly HEPES, sodium bicarbonate, or TRIS) may be used instead of the buffer specifically described. Furthermore, a buffer containing 0.9% NaCl is also useful.

[0093] Optionally, the compositions of the present invention may contain other common pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to rAAV and / or variants and carriers. Suitable representative preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0094] Pharmaceutical compositions containing at least one replication-deficient rAAV virus as described herein may be prepared using physiologically acceptable carriers, diluents, excipients, and / or additives for use in gene transfer and gene therapy applications. In the case of AAV viral vectors, the quantification of genome copies ("GC"), vector genome ("VG"), or viral particles may be used as a measure of the dose contained in the preparation or suspension. Any method known in the art may be used to determine the genome copy (GC) number of the replication-deficient viral composition of the present invention. One method for titrating AAV GC number is as follows: A purified AAV vector sample is first treated with DNase to remove AAV genomic DNA or contaminating plasmid DNA that is not packaged in the capsid from the production process. The DNase-resistant particles are then subjected to heat treatment to release the genome from the capsid. The released genome is then quantified by real-time PCR using a primer / probe set targeting a specific region of the viral genome (usually a poly-A signal). Alternatively, an effective dose of recombinant adeno-associated virus containing a nucleic acid sequence encoding an optimized REP-1 or CNGA3 transgene is determined as described in SKMcLaughlin et al., 1988 J. Virol., 62:1963, which is incorporated in its entirety herein by reference.

[0095] As used herein, the term “dose” may refer to the total dose or the amount delivered to a subject during the course of treatment in a single unit (or multiple units or split dosage) administration. The pharmaceutical viral composition is contained within approximately 1.0 × 10¹⁶ units, including all integers or fractional quantities within the specified range. 9 The number of GCs is approximately 1.0 × 10⁻¹⁰. 15 Individual GC The dosage unit may be prepared to contain a replication-deficient virus having a codon-optimized nucleic acid sequence encoding REP-1, CNGA3, or CNGB3 as described in the present invention in an amount within the specified range. In one embodiment, the composition contains at least 1 × 10⁶ per dose, with all integers or fractional quantities within the specified range.9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 or 9×10 9 prepared to contain . In another embodiment, the composition contains all integer or fractional amounts within the range and at least 1×10 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 or 9×10 10 prepared to contain 11 , 2×10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 or 9×10 11 prepared to contain 12 , 2×10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 or 9×10 12 prepared to contain 13 , 2×10 13 , 3×10 13 , 4×10 13 , 5×10 13 ​​​​, 6×10 13 , 7×10 13 , 8×10 13 or 9 x 10 13 It is prepared to contain GCs. In another embodiment, the composition contains all integers or fractional quantities within the range, with at least 1 × 10 per dose. 14 , 2×10 14 , 3 x 10 14 , 4×10 14 , 5×10 14 , 6×10 14 , 7×10 14 , 8×10 14 or 9 x 10 14 It is prepared to contain GCs. In another embodiment, the composition contains all integers or fractional quantities within the range, with at least 1 × 10 per dose. 15 , 2×10 15 , 3 x 10 15 , 4×10 15 , 5×10 15 , 6×10 15 , 7×10 15 , 8×10 15 or 9 x 10 15 Prepared to contain GCs. In one embodiment, for human application, the dose is 1 × 10 per dose, including all integers or fractional quantities within the range. 10 ~Approx. 1×10 12 This may be within the range of individual GCs. All doses are incorporated herein by reference, for example, M. Lock et al., Hum It may be measured by any known method, including oqPCR or digital droplet PCR (ddPCR) as described in Gene Methods. 2014 Apr. 25(2):115-24. Doi: 10.1089 / hgtb. 2013. 131.

[0096] These doses may be administered in carrier, excipient, or buffer preparations of varying volumes ranging from about 25 to about 1000 microliters, including all numbers, depending on the dimensions of the area to be treated, the viral titer used, the route of administration, and the effectiveness of the desired method. In one embodiment, the volume of the carrier, excipient, or buffer is at least about 25 μL. In one embodiment, the volume is about 50 μL. In another embodiment, the volume is about 75 μL. In another embodiment, the volume is about 100 μL. In another embodiment, the volume is about 125 μL. In another embodiment, the volume is about 150 μL. In another embodiment, the volume is about 175 μL. In yet another embodiment, the volume is about 200 μL. In another embodiment, the volume is about 225 μL. In yet another embodiment, the volume is about 250 μL. In yet another embodiment, the volume is about 275 μL. In yet another embodiment, the volume is about 300 μL. In yet another embodiment, the volume is about 325 μL. In yet another embodiment, the volume is about 350 μL. In another embodiment, the volume is approximately 375 μL. In another embodiment, the volume is approximately 400 μL. In another embodiment, the volume is approximately 450 μL. In another embodiment, the volume is approximately 500 μL. In another embodiment, the volume is approximately 550 μL. In another embodiment, the volume is approximately 600 μL. In another embodiment, the volume is approximately 650 μL. In another embodiment, the volume is approximately 700 μL. In another embodiment, the volume is approximately 700 to 1000 μL.

[0097] In one embodiment, for small animal subjects such as mice, at least 1 × 10 in a volume of about 1 μL to about 3 μL. 9 ~Approx. 1×10 11 The virus control with the dose of individual GCs In some cases, a ct may be delivered. For larger veterinary subjects with eyes roughly the same size as human eyes, the relatively larger human doses and volumes mentioned above are useful. For a discussion of practical guidelines for the administration of substances to various veterinary animals, see, for example, Diehl et al., J. Applied Toxicology, 21:15-23 (2001). This document is incorporated herein by reference.

[0098] To reduce the risk of undesirable effects such as toxicity, retinal dysplasia, and detachment, it is preferable to use the lowest effective concentration of the virus or other delivery vehicle. Further doses within this range may be selected by the attending physician, taking into account the subject being treated, preferably the person's physical condition, the subject's age, any specific eye disease, and, if progressive, the extent to which the disease has manifested.

[0099] Another aspect described herein is the risk of having or developing colloideremia. A method for treating, delaying, or halting the progression of blindness in a mammalian subject. In one embodiment, an rAAV having a REP-1 codon-optimized sequence suspended in a physiologically compatible carrier, diluent, excipient, and / or additive may be administered to a desired subject, including a human subject. This method involves administering to a subject requiring any of the following: a nucleic acid sequence, expression cassette, rAAV genome, plasmid, vector, or rAAV vector or a composition containing them. In one embodiment, the composition is delivered subretinally. In another embodiment, the composition is delivered intravitreously. In yet another embodiment, the composition is administered using a combination of administration routes suitable for the treatment of an eye disease, which may include administration via the eyelid vein or via other intravenous or conventional administration routes.

[0100] Another aspect described herein is a method for treating, delaying, or halting the progression of blindness in mammalian subjects who have or are at risk of developing color blindness. In one embodiment, rAAVs containing a CNGA3 or CNGB3 natural, modified, or codon-optimized sequence, preferably suspended in a physiologically compatible carrier, diluent, excipient, and / or additive, may be administered to a desired subject, including a human subject. This method involves administering to a subject who requires any of the following: nucleic acid sequences, expression cassettes, rAAV genomes, plasmids, vectors, or rAAV vectors or compositions containing them. In one embodiment, the composition is delivered subretinally. In another embodiment, the composition is delivered intravitreously. In yet another embodiment, the composition is administered using a combination of administration routes suitable for the treatment of an eye disease, which may include administration via the eyelid vein or via other intravenous or conventional administration routes.

[0101] For use in these methods, the volume and viral titer of each dose are determined individually as further described herein and may be the same as or different from other treatments performed in the same or opposite eye. Dosage, administration or management may be determined by the attending physician as given herein. In one embodiment, the composition is administered in one affected eye in a single dose selected from those listed above. In another embodiment, the composition is administered in both affected eyes simultaneously or sequentially in a single dose selected from those listed above. Sequential administration may mean a time difference of several minutes, several hours, several days, several weeks, or several months between administrations from one eye to the other. In another embodiment, the method involves administering the composition to the eye in two or more doses (e.g., split doses). In another embodiment, multiple infusions are performed in different parts of the same eye. In another embodiment, a second administration of rAAV containing a selected expression cassette (e.g., a CHM-containing cassette) is performed at a later time point. Such a time point may be several weeks, several months, or several years after the first administration. In one embodiment, such a second dose is performed using an rAAV having a different capsid than the rAAV from the first dose. In another embodiment, the rAAVs from the first and second doses have the same capsid.

[0102] In yet another embodiment, the compositions described in the present invention may be delivered in one or more compositions. Optionally, two or more AAVs or viruses may be delivered [see, for example, International Publication No. 2011 / 126808 and International Publication No. 2013 / 049493]. In another embodiment, the viruses may include different replication-deficient viruses (e.g., AAV and adenovirus).

[0103] In some embodiments of the present invention, it is desirable to perform non-invasive retinal imaging and functional studies to identify the regions of rod and cone photoreceptors to be targeted for therapeutic purposes. In these embodiments, clinical diagnostic tests are used to determine the precise location for one or more subretinal injections. These tests are performed using electroretinography depending on the species of the subjects being treated, their physical condition or health, and the dosage. This may include (ERG), visual field testing, confocal scanning laser ophthalmoscopy (cSLO), and topographical mapping of retinal layers and measurement of their thickness by optical coherence tomography (OCT), topographical mapping of cone density via adaptive optics (AO), and functional eye exams. From the standpoint of imaging and functional studies, in some embodiments of the present invention, one or more injections are performed in the same eye to target various areas of the affected eye. The volume and viral titer of each injection are determined individually as further described herein and may be the same as or different from other injections performed in the same or opposite eye. In another embodiment, a single relatively large volume injection is performed to treat the entire eye. In one embodiment, the volume and concentration of the rAAV composition are chosen to affect only the areas of impaired ophthalmic cells. In another embodiment, the volume and / or concentration of the rAAV composition are larger to reach a larger portion of the eye containing unimpaired photoreceptors.

[0104] In one embodiment of the method described herein, a single intraocular delivery of the composition described herein, for example, AAV delivery of an optimized REP-1 cassette, is useful in preventing visual loss and blindness in subjects at risk of developing colloideremia. In another embodiment of the method described herein, a single intraocular delivery of the composition described herein, for example, AAV delivery of an optimized CNGA3 or CNGB3 cassette, is useful in preventing visual loss and blindness in subjects at risk of developing color vision deficiency.

[0105] Thus, in one embodiment, the composition is administered before the onset of the disease. In another embodiment, the composition is administered before the onset of visual impairment or loss. In yet another embodiment, the composition is administered after the onset of visual impairment or loss. In yet another embodiment, the composition is administered when less than 90% of the rods and / or cones or photoreceptors are functioning or remaining compared to an unaffected eye.

[0106] In another embodiment, the method includes conducting additional studies, such as functional and imaging studies to determine the effectiveness of the treatment. For examinations in animals, such tests include assessment of retinal and visual function via electroretinography (ERGs) to observe rod and cone photoreceptor function, visual motor nystagmus, pupillary measurement, water maze test, light-dark preference, optical coherence tomography (for measuring the thickness of various layers of the retina), and histology (retinal thickness, rows of granules (nuclei) in the outer granular layer, immunofluorescence to demonstrate transgene expression, cone photoreceptor counting, and staining of retinal sections with peanut glutenin to identify cone photoreceptor sheaths).

[0107] For human subjects specifically, following administration of the composition in the doses described herein, subjects are examined for the efficacy of the treatment using electroretinography (ERGs) to test rod and cone photoreceptor function, pupillary visual acuity, contrast sensitivity color vision testing, visual field testing (Humphrey / Goldmann visual field), perimetry mobility test (obstacle course), and reading speed test. Other useful post-treatment efficacy studies to which subjects are exposed following treatment with the pharmaceutical composition described herein include functional magnetic resonance imaging (fMRI), full-field light sensitivity testing, retinal structure studies including optical coherence tomography, fundus photography, fundus autofluorescence, adaptive optics laser scanning fundus examination, mobility tests, reading speed and accuracy tests, microperimetry, and / or fundus examination. These and other efficacy studies are incorporated herein by reference as U.S. 8 , Specification No. 147,823; as described in the concurrently pending international patent application publications, International Publication No. 2014 / 011210 or International Publication No. 2014 / 124282.

[0108] In yet another embodiment, any of the above methods may be combined with another or secondary treatment. In yet another embodiment, a method of treating these eye diseases may involve treating a subject with a combination of the compositions described in detail herein with another treatment, such as an antibiotic treatment or an analgesic treatment. The additional treatment may be any known or yet unknown treatment that helps prevent, halt or improve these mutations or defects or any effects associated therewith. Secondary treatment may be administered before, concurrently with, or after the administration of the above compositions. In one embodiment, secondary treatment may include nonspecific methods for maintaining the health of retinal cells, such as the administration of neurotrophic factors, antioxidants, or anti-apoptosis agents. Nonspecific methods may be achieved through the injection of proteins, recombinant DNA, recombinant viral vectors, stem cells, fetal tissue, or genetically modified cells. The latter may include encapsulated genetically modified cells.

[0109] In one embodiment, a method for producing recombinant rAAV comprises obtaining a plasmid containing the above-described AAV expression cassette and culturing packaging cells possessing the plasmid in the presence of sufficient viral sequences to enable packaging of the AAV viral genome within an infectious AAV envelope or capsid. Specific methods for producing rAAV vectors are described above and may be used to produce rAAV vectors capable of delivering codon-optimized REP-1, CNGA3, or CNGB3 in the expression cassette and genome described in the above and below examples.

[0110] In yet another embodiment, we provide a vector comprising one of the expression cassettes described herein. As described above, such vectors can be plasmids of diverse origins and are useful in some embodiments for the production of recombinant replication-deficient viruses, as further described herein.

[0111] In one alternative embodiment, the vector is a plasmid containing an expression cassette, where the expression cassette contains an AAV inverted terminal repeat sequence and a codon-optimized nucleic acid sequence encoding REP-1 and an expression regulatory sequence that directs the expression of the encoded protein in the host cell.

[0112] In another embodiment, the vector is a plasmid containing an expression cassette, where the expression cassette contains an AAV inverted terminal repeat sequence and a codon-optimized nucleic acid sequence encoding CNGA3 and an expression regulatory sequence that directs the expression of the encoded protein in the host cell.

[0113] In another embodiment, the vector is a plasmid containing an AAV expression cassette, where the expression cassette includes an AAV inverted terminal repeat sequence and a codon-optimized nucleic acid sequence encoding CNGB3 and an expression regulatory sequence that directs the expression of the encoded protein in the host cell.

[0114] It should be noted that the terms “a” or “an” refer to one or more. Therefore, “a” (or “an”), “one or more” and “at least one” are used interchangeably herein.

[0115] The terms “comprise”, “comprises”, and “comprising” should be interpreted inclusively, not exclusively. The term “comprising” and its variations should be interpreted exclusively, not inclusively. While various aspects in the specification are presented using the language of “comprising,” it is also intended and explained that, under other circumstances, the relevant aspects should be interpreted using the language of “consisting of” or “consisting essentially of.”

[0116] As used herein, the term “about” means a 10% variation from a given standard unless otherwise specified.

[0117] The term "modulate" or its variation thereof, as used herein, refers to the ability of a composition to inhibit one or more components of a biological pathway.

[0118] Unless otherwise defined herein, the technical and scientific terms used herein have the same meaning as they would normally be understood by a person of ordinary skill in the art and by reference to published books, which provide a general guide to many of the terms used herein. [Examples]

[0119] The following examples are illustrative and not intended to limit the present invention.

[0120] Example 1 - Differentiation of pluripotent stem cells into RPE Colloideremia lacks a relevant mouse model, and there is no canine model; therefore, we will investigate transduction and expression in a human retinal cell model of the disease. Since it is impossible to obtain retinal cells from living subjects, RPEs will be generated from induced pluripotent stem cells. Pluripotent stem cells will be directed to RPEs using the protocol described by Buchholz et al., Rapid and Efficient Directed Differentiation of Human Pluripotent Stem Cell Into Retinal Pigmented Epithelium, Stem Cells Translational Medicine, 2013;2:384-393, which is incorporated entirely herein by reference. Cereso et al., Proof of concept for AAV2 / 5-mediated gene therapy in iPSC-derived retinal See also pigment epithelium of a choroideremia patient, Molecular Therapy-Methods & Clinical Development (2014) 1, 14011. Other methods for preparing RPE are known in the art.

[0121] In short, human induced pluripotent stem cell lines are maintained on a mitomycin C-treated or irradiated mouse embryonic fibroblast support cell layer in Dulbecco's modified Eagle's medium: Nutrient Mixture F-12 (DMEM / F12) containing 2 mM GlutaMAX-I, 20% knockout serum replacement, 0.1 mM Modified Eagle's Medium Non-Essential Amino Acids (MEM NEAA), 0.1 mM β-mercaptoethanol, and 4 ng / ml bFGF.

[0122] Pluripotent stem cells are directly subcultured on Matrigel (BD Biosciences) in DMEM / F12 containing IX B27, IX N2, and IX NEAA (Invitrogen). From day 0 to day 2, 50 ng / ml Noggin and 10 ng / ml Add Dkk1, 10 ng / ml IGF1, and 10 mM nicotinamide to the basic culture medium. From day 2 to day 4, add 10 ng / ml Noggin, 10 ng / ml Dkk1, 10 ng / ml IGF1, 5 ng / ml bFGF, and 10 mM nicotinamide to the basal medium. From day 4 to day 6, add 10 ng / ml Dkk1, 10 ng / ml IGF1, and 100 ng / ml Activin A (R&D Systems) to the basal medium. From day 6 to day 14, add 100 ng / ml Activin A, 10 μM SU5402 (EMD Millipore, Darmstadt, Germany), and 1 mM VIP to the basal medium. Perform control standard experiments in basal medium only (DMEM / F12, B27, N2, and NEAA).

[0123] Cells are mechanically concentrated by scraping off cells with non-RPE morphology. The remaining RPE is then digested using TrypLE Express (Invitrogen) at 37°C for 5 minutes. The cells are passed through a 30-μm single-cell strainer and seeded onto a Matrigel-coated tissue culture plastic Transwell membrane or a CC2-treated chamber slide. The concentrated cells are cultured for 30 days in DMEM-high glucose containing 1% fetal bovine serum (FBS), GlutaMAX, and sodium pyruvate.

[0124] Example 2 - Cells transduced using AAV-REP-1 In short, an AAV2 / 8CMV.CBA-REP-1 viral vector with an optimized REP-1 codon sequence is prepared by transient transfection of HEK293 cells, and the viral particles are precipitated from either supernatant using polyethylene glycol. See, for example, Guo et al., Rapid and simplified purification of recombinant adeno-associated virus, J. Virol Methods. 2012 Aug;183(2):139-146, which is incorporated herein by reference. The vector is purified by double CsCl centrifugation, dialyzed, and titrated by dot blot assay.

[0125] For the prenylation experiment, RPE was seeded in a 24-well plate, and 1.2 × 10⁶ 6 pieces The number of cells is estimated to be in a confluent state. The cells are transduced using 100,000 vector genomes (vg) per cell, and a prenylation assay is performed 4 weeks after transduction. The experiment is repeated three times under the same conditions.

[0126] Example 3 - Prenylation As described above in Vasireddy et al., PloS One. 2013 May 7;8(5):e61396, an in vitro prenylation assay was performed using [3H]-geranylgeranyl pyrophosphate (GGPP) (Perkin Elmer, Boston, MA, USA) as the prenylation group donor, in the presence of recombinant Rab geranylgeranyltransferase and RAB27. The introduction of radiolabeled prenylation groups into the RAB27 protein was measured using a scintillation counter. For consistency, the control standard value was normalized to 100 and used as the baseline value. All experiments were repeated three times, and a statistical comparison of prenylation between the experimental group and the control standard group was performed using a two-tailed unpaired student test. Evaluation will be performed using the student's test.

[0127] In short, transduced REP cells are washed with cold PBS 48 hours after transduction. The cell pellet is collected and thoroughly washed with cold PBS. The cells are lysed on ice for 30 minutes using RIPA + protease inhibitors. In another protocol, the cells are sonicated. The cytoplasm is extracted by centrifugation of the cell lysate at 75,000–100,000 g for 1–2 hours at 4°C. Collect the images.

[0128] Prepare the stock solution for the prenylation reaction as follows.

[0129] [Table 1]

[0130] The final reaction volume used for prenylation is 25 μL.

[0131] [Table 2]

[0132] Incubate the reaction mixture at 37°C for 30 minutes. Stop the reaction by adding 9:1 ethanol / HCl and incubate for another 30 minutes. Collect the protein on glass fiber filter paper (Whatman papers) by vacuum filtration (0.1 ml). Carefully wash the filter three times with cold phosphate buffer to remove unbound material. Carefully dry the membrane. Place the filter in 5 ml of scintillation cocktail and perform scintillation counting. See also Tolmachova et al., CHM / REP1 cDNA delivery by lentiviral vectors provides functional expression of the transgene in the retinal pigment epithelium of choroideremia mice, The Journal of Gene Medicine, 2012; 14-158-68, which is incorporated in its entirety herein by reference.

[0133] Assays for proof-of-concept of CNGA3 or CNGB3 may involve the use of naturally occurring mutant animal models (e.g., Cnga3- / - mice or Awassi sheep). The mouse models can be housed together with Nrl- / - mice, which are "all-cone" photoreceptor mice, to obtain double knockouts. The latter (Cnga3- / -Nrl- / -) mice may expedite the identification of efficacy. Efficacy can be measured by pupillary measurements (e.g., using optokinetics), electroretinography, and visual behavior, which are measures of visual acuity and contrast. Ultimately, histology will demonstrate transgene expression with improved results compared to other measures. Histological methods may also be used to demonstrate the effect of intervention on cone photoreceptors (e.g., total number, density, and location of cone photoreceptors).

[0134] Similar to the colloideremia discussed above, assays for proof-of-concept regarding gene augmentation therapy for CNGA3- or CNGB3-associated color blindness may involve the use of induced pluripotent stem cell (iPSC) models. iPSC models created from subjects with color blindness due to CNGA3 or CNGB3 mutations are differentiated in vitro into retinal precursor and / or photoreceptor cells. These cells are delivered with wild-type CNGA3 (or CNGB3) cDNA using recombinant AAV, and the cells are analyzed for conservation of biosynthesis and function of the associated (cyclic nucleotide-sensitive, CNG) channels composed of these subunits. Channel function is assessed by electrophysiology of membrane patches. Channel restoration should rescue the cGMP-activating current. Additional studies can investigate the sensitivity of channel function before and after delivery of wild-type CNG cDNA to physiological ligands.

[0135] Example 4: In vitro expression of AAV. codon-optimized human CHM The objective of this study was to evaluate the ability of AAV-mediated CHM expression in 84-31 and COS-7 cell lines after gene delivery using a series of next-generation AAV2 and AAV8 vectors encoding the codon-optimized CHM gene (SEQ ID NO: 1).

[0136] To maximize CHM expression, a codon-optimized CHM sequence was constructed (SEQ ID NO: 1). The codon-optimized plasmid was synthesized and used in the construction of all next-generation CHM transgene expression cassettes. To overcome the potential problem of non-functional AAV genome contamination, a non-coding lambda stuffer region was included in the vector backbone. The introduction of the stuffer not only increases plasmid length but also reduces the possibility of plasmid DNA backbone contamination during AAV packaging. The effect of introducing the stuffer region in the vector backbone to remove plasmid DNA impurities was studied independently. Various constructs were constructed using two recombinant AAV provirus plasmids (high-copy and low-copy) backbone. The high-copy plasmid was designed based on a pUC vector origin. The low-copy plasmid was designed based on a p15A origin. To further enhance translation from the correct start codon, a Kosack sequence was introduced upstream of the start codon.

[0137] A total of four plasmids were created for this study and the studies described in the following examples (Table 1). In addition, a plasmid containing the CHM natural sequence currently used in clinical trials was also created (version 1). Plasmid maps for versions 2a, 2b, 3a, and 3b, as well as version 1, are shown in Figure 6-10.

[0138] [Table 3]

[0139] The in vitro expression of these constructs was investigated in COS-7 and 84-31 cell lines. The manipulated characteristics of the next-generation CHM constructs are described in Table 1.

[0140] Recombinant AAV provirus high-copy and low-copy plasmids were constructed by cloning codon-optimized human CHM cDNA (hCHM) (SEQ ID NO: 1) into a transgene cassette. The transgene was placed under the regulation of a hybrid chicken β-actin (CBA) promoter. This promoter consists of a cytomegalovirus (CMV) early enhancer, a proximal chicken β-actin promoter, and CBA exon 1 flanked by an intron 1 sequence. The provirus high-copy and low-copy plasmids also contain AAV inverted terminal repeat sequences and poly(A) sequences. The next-generation plasmid backbone used in the current study contains a lambda phage fragment stuffer and a kanamycin bacterial selection gene. The gene follows. The additional plasmid lacks a stuffer but contains the kanamycin selector gene. The high-copy-number vector is similar to that of the pUC plasmid (approximately 300 copies per bacterial cell). The low-copy-number plasmid (approximately 10 copies per bacterial cell) originates from p15A. To enhance translation from the correct start codon, all next-generation constructs contain a Kozak common sequence upstream of the start codon, ATG. The constructed plasmids are sequence-validated using primers that can specifically target either the promoter + enhancer extension sequence or the codon-optimized CHM sequence. Plasmid maps and sequences of all five constructs are shown in Figure 6-10. The AAV vectors listed below were constructed using standard triple transfection with calcium phosphate (see Table 2 for vector qualification). Vectors for both AAV2 and AAV8 serotypes were constructed to confirm that the results were serotype-independent.

[0141] [Table 4]

[0142] The 84-31 cell line is a subclone of the 293HEK cell line (human embryonic kidney cells) that constitutively expresses adenovirus E4 protein to enhance AAV virus transduction. The COS-7 cell line is a fibroblast-like cell line derived from monkey kidney tissue. Both 84-31 and COS-7 cells were cultured separately in 6-well cell culture plates and transduced using one of 10 test articles (either AAV2 or AAV8) at five different multiplicities of infection (MOIs). After 36–48 hours, cells were harvested, lysed, and protein samples were prepared for SDS-PAGE and subsequent Western blot analysis to detect exogenous CHM expression.

[0143] In an environment supplied with 5% CO2, at 37°C, 10% fetal bovine serum and 1% penicillin were used. Both 84-31 and COS-7 cells were cultured in Dulbecco's modified Eagle medium (DMEM)-high glucose containing syrin / streptomycin. 3 × 10⁶ cells were cultured the day before transduction (18-24 hours prior). 5 Cells at a density of (3E5) - Cells were seeded in 2 ml of cell medium in each well of a well-type cell culture dish. The seeded cells were incubated at 37°C in an environment supplied with 5% CO2. COS-7 and 8 Both wells of 4-31 cells were infected with the AAV vectors listed below at various modalities of infection (MOI) (Tables 3 and 4). Negative control standard cells (cells not transduced) were not treated with the virus. In short, the tissue medium was removed, and aliquots of 2 ml of fresh medium were added to each well of the 6-well culture dish. Then, a predetermined amount of AAV vector was measured (directly from the stock solution) and added to each well (Tables 3 and 4). 1 × 10⁻⁶ 4 MO For I, 1 μL of each viral stock solution was diluted to 10 μL in cell culture medium. From this solution, a predetermined volume of virus was added to each well (Tables 3 and 4). Cells were incubated at 37°C with 5% CO2 for 36–48 hours until the AAV virus was recovered. The cells were incubated. Before harvesting, the cells were observed under a microscope for testing for abnormalities.

[0144] [Table 5]

[0145] [Table 6]

[0146] First, we investigated whether the in vitro expression of CHM was cell line independent using both the COS7 and 84-31 cell lines. Once independence was confirmed, all subsequent experiments were performed only in 84-31 cells, which showed superior transduction efficiency using AAV. Wells of 84-31 cells were infected with the AAV vectors listed below at various MOIs (see Tables 3 and 4).

[0147] Western blot analysis: 1. Cell lysates were prepared. 36-48 hours after infection, AAV-transduced cells were collected along with untreated control standard cells after thorough PBS washing. The cells were then lysed on ice with RIPA buffer containing a protease inhibitor. The cell lysate was cleared by centrifugation at 13,000 rpm for 10 minutes. 2. Protein quantification and preparation. Protein quantification of the cell lysate was performed using the ThermoFisher Micro BCATM Protein Assay Kit according to the manufacturer's instructions. Protein concentration was determined by obtaining the OD reading at 562 nm. To evaluate the in vitro expression of CHM, 40-60 μg of the measured protein was loaded onto 4-12% Bis-Tris gels. 3. SDS-PAGE and blotting SDS-PAGE and Western blot analysis were performed according to known protocols. In short, the protein gel was transferred to a nitrocellulose membrane, blocked in milk, and incubated with the primary antibody. Anti-human REP-1 2F1 antibody (2F1, 1:1000 dilution) and one of the following: anti-GAPDH antibody (1:1000 dilution), anti-actin antibody (1:1000 dilution), or anti-tubulin antibody (1:5000 dilution) were used as primary antibodies for each blot. After washing the blots, HRP-conjugated anti-mouse IgG antibody and / or anti-rabbit IgG antibody at a concentration of 1:5000 were used as secondary antibodies. The blots were chemiluminescent using ECL reagent according to the manufacturer's instructions. Control Standards: 1. Loading Control Standards: One of the following: anti-actin antibody, anti-tubulin antibody, or anti-GAPDH antibody was used as a loading control standard to indicate equal protein loading in each well of the gel. Anti-tubulin antibody detects proteins of ~51 kDa, anti-actin antibody detects proteins of ~42 kDa, and anti-GAPDH antibody detects proteins of ~39 kDa. The initial blot was examined (probed) using either an anti-tubulin antibody, an anti-actin antibody, or an anti-GAPDH antibody, depending on their availability. After the initial experiment, for consistency, the anti-GAPDH antibody was used as a loading control standard.2. Positive control standard: After confirming hREP-1 protein production in AAV2.V2a-transduced COS-7 cells, AAV2.V2a-Cos-7 cell lysate was used as the positive control standard in subsequent Western blotting experiments. 3. Negative control standard: Untreated cells were used as the negative control standard. Table 5 summarizes the analysis of Western blotting results for REP-1 protein production in various cell lines.

[0148] [Table 7]

[0149] Monoclonal human REP-1-specific antibodies detected a single ~75-80 kDa hREP-1 protein in cells transduced using next-generation AAV2.copt.CHM / AAV8.copt.CHM. No 75-80 kDa band was observed in cell lysates of untreated control standard cells. Blut exploration using any of the anti-actin / anti-tubulin / anti-GAPDH antibodies showed bands of equal concentration in all columns of the Western blot, including the untreated control standard. The anti-actin antibody detected a protein molecular weight band at ~42 kDa, the anti-tubulin antibody detected the protein at ~51 kDa, and the anti-GAPDH antibody detected the protein at ~39 kDa. All antibodies detected only specific bands within the predicted size molecular weight range. No nonspecific bands were observed in any of the blots. The molecular weight of the protein in question was compared using pre-stained molecular weight markers.

[0150] In summary, the REP-1 protein was observed at the predicted size in COS-7 and 84-31 cells transduced using AAV2.V2a, AAV2.V2b, AAV2.hCHM.V3a, and AAV2.hCHM.V3b. Untreated control standards did not reveal the presence of human REP-1 protein of the predicted size. Labeling of blots with anti-actin antibodies detected protein bands of the same intensity at ~42 kDa in all columns of the gel. The molecular weights of REP-1 and actin were compared using a pre-stained protein ladder. Data are not shown.

[0151] The results indicate that AAV2 and AAV8 serotype vectors containing next-generation plasmids can effectively transduce 84-31 and COS-7 cells. CHM expression in the next-generation plasmids was within the detectable range and showed a dose-dependent trend. Transduction of cells using the next-generation hCHM virus resulted in the predicted size of REP-1 protein. It led to production.

[0152] Example 5: Comparison of in vitro protein expression between AAV codon-optimized human CHM and AAV natural human CHM. The objective of this study was to describe (delineate) the transduction efficiency of AAV vectors (serotypes 2 and 8), including various versions of the CHM-containing transgene cassette, by measuring the level of REP-1 protein in a study model based on the 84-31 cell line.

[0153] Plasmids and Vectors: A total of five transgene plasmids were compared in either AAV2 or AAV8: Version 1 (previously used in ongoing clinical trials) and four next-generation versions (V2a, V2b, V3a, and V3b). Plasmids were constructed as described in Example 4, and their characteristics are shown in Table 1. Table 2 above summarizes the AAV2 and AAV8 vectors produced and the concentrations of the viral stock solutions.

[0154] Research design (e.g., processing groups) 1. In a pilot experiment, COS-7 and 84-31 cells were transduced using AAV2.hCHM. versions 1, 2a, and 2b. Western blotting was performed to compare the transduction efficiency levels in the two cell lines.

[0155] 2.10 One of the test substances (versions 1, 2a, 2b, 3a, and 3b in either AAV2 or AAV8 background) 3 × 10 5 Used in MOI, in a 6-well plate 84–31 cells cultured in [specific medium] were transduced. After 36–48 hours, the cells were harvested and lysed. The cell lysates were loaded onto SDS-PAGE plates and subjected to further Western blot analysis. REP-1 protein levels were compared among all construct versions. Two separate plates were prepared for each AAV2.CHM or AAV8.CHM experiment and analyzed individually.

[0156] Test material administration 3.4.1 Cell culture At 37°C in an environment supplied with 5% CO2, 10% fetal bovine serum and 1% penicillin were used. Both 84-31 cells and COS-7 cells were cultured in Dulbecco's modified Eagle medium (DMEM)-high glucose containing phosphorus / streptomycin.

[0157] 3.4.2 Preparation of cells for transduction The day before transduction (18-24 hours prior), 84-31 and COS-7 cells were placed in 2 ml of cell medium per well of a 6-well cell culture dish, 3 × 10⁶ cells per well. 5 Seeds were sown at a density of (3E5). The seeded cells were incubated at 37°C in an environment supplied with 5% CO2. .

[0158] 3.4.3 Trait introduction Place 3 × 10⁶ cells and COS-7 wells into the AAV vector described below. 5Infection was carried out at the MOI (Table 6 for the pilot experiment, and for the second group of experiments). See Table 7). No virus was added to the negative (untransduced) control standard. In short, the tissue medium was first removed from each well of the 6-well cell culture dish and replaced with 2 ml of fresh medium per well. Then, a predetermined amount of AAV vector (see Table 2 for the vector volume used for transduction) was measured (from stock solution) and added directly to each well. Cells were AAV-transduced at 37°C using 5% CO2. The cells were incubated with the virus for 36-48 hours until collection. Before collection, the cells were observed under a microscope for abnormality testing. Western blotting was performed as described in Example 4. An analysis was performed.

[0159] [Table 8]

[0160] [Table 9]

[0161] Results: Comparison of expression of native hCHM (AAV2.hCHM.V1) versus codon-optimized CHM AAV2a and 2b versions in 84-31 and COS-7 cells. In this experiment, 84-31 and COS-7 cells were transduced using AAV2.hCHM. version 1, AAV2.hCHM. version 2a, or AAV2.hCHM. version 2b without a vector (untreated control standard). Western blot analysis using anti-human REP-1 antibody showed that REP-1 protein levels were detectable at ~75-80 kDa in all AAV2 (V1, V2a, V2b) transduced samples and in both cell lines (data not shown). Slightly superior protein expression was observed in the 84-31 cell line (Table 8). Anti-REP1 antibody detected negligible amounts of REP-1 protein in untreated cells. Blot labeling using GAPDH antibody detected a band at ~39 kDa in all cell lysates, including untreated cells.

[0162] Concentration-measuring evaluation of blots using ImageJ software (quantification of protein levels), after normalizing values ​​to endogenous GAPDH protein expression, showed that transduction efficiency was similar in 84-31 and COS-7 cells. (See Table 8 for results). Based on this, the 84-31 cell line, which is of human origin, was used for further experiments.

[0163] Ultimately, AAV2.V1, AAV2.V2a, and Aav2.V2b induced REP-1 protein production in both 84-31 and COS-7 cells with similar transduction efficiencies.

[0164] [Table 10]

[0165] Comparison of expression of native CHM versus codon-optimized CHM AAV2 vectors in 84-31 cells: Western blot analysis of 84-31 cells transduced with AAV2.hCHM.V2a, V3a, V2b, V3b, and V1 using anti-human REP-1 antibody detected bands at ~75-80 kDa under all conditions (data not shown). Anti-REP1 antibody detected negligible amounts of REP-1 protein in untreated cells. Labeling of blots with GAPDH antibody detected bands at ~39 kDa in all cell lysates, including untreated cells. Concentration-metric evaluation of blots using ImageJ software (quantification of expression levels), after normalizing values ​​to endogenous GAPDH protein production, showed increased expression of AAV2.hCHM.V2a, 3a, 2b, and 3b compared to AAV2.hCHM.V1. See Tables 9 and 10 for results.

[0166] [Table 11]

[0167] [Table 12]

[0168] Comparison of codon-optimized CHM expression in native CHM versus AAV8.V1, V2a, V3a, V2b, V3b vectors in 84-31 cells: Western blot analysis of cells transduced using AAV8.V1, AAV8.V2a, AAV8.V3a, AAV8.2b, and AAV8.3b detected a band at ~75-80 kDa in all transduced cells (data not shown). The anti-REP1 antibody detected negligible amounts of REP-1 protein in untreated cells. Labeling of the blot with GAPDH antibody detected a band at ~39 kDa in all cell lysates, including untreated cells. Concentration-metric evaluation of the blot using ImageJ software showed higher expression of AAV8.hCHM.V2a;3a;2b;3b compared to AAV8.V1. The values ​​were obtained by first normalizing the CHM values ​​to the expression level of each endogenous GAPDH protein, and then normalizing them to the mean expression level of version 1. See Tables 11 and 12 for the results.

[0169] [Table 13]

[0170] [Table 14]

[0171] Conclusion: Comparative expression studies showed that in 84-31 cells, application of AAV vectors containing next-generation AAV.hCHM. versions 2a, 2b, 3a, and 3b induced increased REP-1 protein production in both AAV2 and AAV8 serotype vectors compared to version 1 (currently used in clinical trials).

[0172] Example 6: Evaluation of the effect of lambda stuffer on AAV vector production by qPCR titer analysis. To evaluate the effect of lambda stuffer sequences on the amount of DNA impurities, a single qPCR (quantitative polymerase chain reaction) experiment was performed for all eight AAV vectors shown in Table 2 above. Linearized AAV plasmid standards were used to prepare assay standards. Primer-probe sets were designed on either the CMV / CBA promoter region for quantification of correctly packaged AAV genomes or the kanamycin resistance (KanR) coding region for reverse packaging. Standards and vector samples were experimented with using one CMV / CBA primer-probe set and two other sets using KanR sets. Vector sample values ​​(viral genome copies per mL) were determined from each standard curve. The effect of stuffer sequences was evaluated by comparing the relative amounts of KanR-containing impurities in each vector lot with respect to the CMV / CBA-containing sequence. Reagents: Transgene-containing viral vector titer: Reference standard: CMV-CBA promoter Primer: CMV-F: CCC ACT TGG CAG TAC ATC AA CMV-R:GCC AAG TAF GAA AGT CCC ATA A FAM-Probe: / 56-FAM / CA TAA TGC C / ZEN / A GGC GGG CCA TTT AC / 3IABkFQ / Impurities - contained viral vector titer: Reference standard: Kanamycin resistance gene Primer: KAN-F:GAT GGT CGG AAG TGG CAT AA KAN-R:TGC GCC AGA GTT GTT TCT FAM-Probe: / 56-FAM / CC GTC AGC C / ZEN / A GTT TAG TCT GAC CA / 3IABkFQ / Dilution reagents: Diluent Q (0.001% PF-68 in nuclease-free water): Dilute 1% PF-68 solution 1000-fold with sterile water. Diluent S: Diluent Q + 2 ng / μL salmon sperm DNA (Agilent Technologies Cat#201190). ABI TaqMan TM Universal Master Mix (Applied Biosystems 4304437 / 4326708) Quiagen PCR Product Purification Kit(Quiagen 28104) ·ABI QuantStudio 6 Flex System

[0173] Sample preparation Below: 5 μL of Dnase buffer (10X), 30 μL of nuclease-free H2O, A DNase digestion solution was prepared by mixing 5 μL of DNase I (Invitrogen, 18068-015).

[0174] Each AAV vector sample was mixed in 10 μL and incubated at ambient temperature for 10 minutes. The digestion mixture was inactivated by adding 50 μL of SDS / EDTA / NaCl solution (0.2% SDS / 5 mM EDTA / 0.2 M NaCl), and incubated at 95°C for 10 minutes. Each AAV vector sample was diluted 10-100,000 times in diluent S for qPCR analysis.

[0175] qPCR standard preparation Reference standard DNA (linearized) was prepared by digesting plasmid p1008 (a low-copy transgene plasmid without a stuffer) using XhoI and purifying it using the Quiagen PCR purification kit. The purified material was identified by analysis on an agarose gel and quantified using Nanodrop. The following equation (equivalence): 1 bp = 1.096 × 10⁻¹⁶ -21 The DNA copy number was determined from the original solution concentration using g. qP The CR standards were prepared according to the following table:

[0176] [Table 15]

[0177] PCR reaction setup The extracted DNA samples were analyzed in a single qPCR experiment, with the same conditions repeated three times (three wells). The experiment included a reference DNA standard in the range of 10³–10⁸ copies per well, and the same conditions were repeated three times. A no-template control (NTC) was included as a negative control standard. Each AAV vector preparation was analyzed using both CMV / CBA and KanR primer / probe sets. Similarly, for quantification of each set, the standards were also analyzed using both CMV / CBA and KanR primer / probe sets.

[0178] [Table 16]

[0179] The PCR reaction conditions were set as follows: 50°C for 2 minutes, 1 cycle; 95°C for 10 minutes. 1 cycle: 95°C, 15 seconds; 40 cycles: 60°C, 1 minute; 40 cycles Experimental Performance. Standards were prepared and tested with 10³–10⁸ DNA copies per well. Since assay sensitivity was not a critical factor for this experiment, the lower limit of the assay was set to 1000 copies. A standard curve for the experiment was created using the standard copy number and standard threshold cycle (CT) values. Linear regression of the standard was performed using ABI software (data not shown). The standard curve fit showed a correlation coefficient (R² value) of 0.998 or greater, indicating a reliable fit model. The slope of the standard curve was -3.5. The efficiency of the amplification reaction was calculated using the slope, and values ​​between -3.2 and -3.6 indicated amplification efficiencies of 90%–110%. Both standard reactions were performed at high rates of 92.6–93.8% (run). The precision of the triple wells ranged from 2–10%, showing excellent agreement between replicated products. The template-less control standard (NTC) produced unquantifiable amplification below the lower limit of the assay.

[0180] [Table 17]

[0181] result: Sample Value Determination: Sample values ​​(AAV genome and reverse-packaging copy number) were interpolated from each fitted standard curve (CMV / CBA or KanR) using CT values. The interpolated DNA copy numbers were corrected for initial dilution and / or digestion dilution. An additional correction factor of 2 was applied to account for the difference between double-stranded DNA standards and single-stranded DNA in the sample.

[0182] The results of the analysis of eight AAV vectors are summarized in the table below, along with quantitative comparisons between the transgene-containing AAV concentration (CMV / CBA) and the KanR-containing impurity concentration. The analysis of the results shows that insertion of lambda stuffers into the transgene effectively reduces the occurrence of plasmid-backbone DNA (i.e., KanR) packaging during AAV production by ~7-20 times (Figure 11).

[0183] [Table 18]

[0184] Example 6: In vitro expression of next-generation AAV8 vector in iPS cells by Western blotting The objective of this study was to evaluate the ability of AAV-mediated CHM expression in induced pluripotent cell lines (iPSCs) after gene delivery using a series of next-generation AAV2 and AAV8 vectors containing codon-optimized REP-1 coding genes.

[0185] Induced pluripotent stem (iPS) cell methods have been successfully used as a platform for testing gene therapy vectors in several proof-of-concept and gene therapy studies, including those for ocular diseases. These subject-specific iPS cells provide a useful in vitro model system for studying disease pathogenesis and establishing models for proof-of-concept testing of gene therapy when relevant animal models are unavailable. As a preliminary step to testing our AAV-mediated gene enhancement therapy for colloideremia (CHM), we generated iPS cells from human subjects with mutations in the causative gene CHM encoding Rab escort protein 1 (REP-1) (see Example 1) (methods are described in NCP.003). We evaluated the in vitro expression of our next-generation AAV.codon-optimized.CHM construct using the generated iPS cells.

[0186] The plasmids and vectors were as described in Example 4. Induced pluripotent stem (iPS) cells are laboratory-generated stem cells from somatic cells, peripheral blood mononuclear cells, which are then reprogrammed to return to a pluripotent state. Reprogramming of blood cells enables the development of personalized in vitro cell models for therapeutic applications. In this report, iPS cells from subjects affected by CHM were used to investigate the in vitro endogenous production of the REP-1 protein via Western blot analysis. The following table (Table 17) describes the details of the iPS cells studied and the respective mutations that cause CHM disease.

[0187] [Table 19]

[0188] Research design (e.g., processing groups) 1.12 well iPS cells cultured on a 12-well cell culture plate are transferred to AAV2.hCHM version 1, version 2a; version 2b; version 3a; version 3b (AAV2.V1;V2a;V2b;V3a;V3b) in 1 × 10⁶ 5 or 3 × 10 5 Infection was performed at one of the MOIs. After 24 hours of transduction, 1 ml of iPS cell medium was added to the cells. After 36–48 hours of transduction, the cells were harvested, lysed, and processed for SDS-PAGE and subsequent Western blot analysis. REP-1 protein production in transduced cells was evaluated using all versions of the construct and compared to untreated control standards.

[0189] 2. As a pilot experiment, three iPS cell lines cultured on 12-well cell culture plates were subjected to 1 × 10⁶ sampling using AAV8.hCHM version 1 and AAV8.hCHM version 2a (AAV8.V1; AAV8.V2a). 6 Transduction is performed using the MOI of iPS cells. Cell lines were derived from three CHM-affected subjects with unrelated mutations in the REP1 gene and were cultured on separate plates for this purpose. After 36–48 hours, cells were harvested, lysed, and subjected to Western blot analysis compared to untreated cell lysates.

[0190] Test material administration 3.4.1 Cell culture iPS cell culture from CHM subjects. In short, iPS cells were cultured on mouse embryonic fibroblasts (MEFs, supporting cells) in iPS cell culture medium at 37°C in an environment supplied with 5% CO2 and 5% O2.

[0191] 3.4.2 Preparation of cells for transduction The day before seeding the cells, 12-well dishes were coated with Matrigel as described in reference NCP.003 (NCP.003: Culturing of iPS cells from CHM patient and controls). Before transducing iPS cells using either AAV2 or AAV8 viral vectors, cells cultured on MEFs were seeded onto Matrigel without MEFs (culture without supporting cells). 4.5 × 10⁶ cells were added to 1 ml of iPS cell medium in each well of the 12-well cell culture dish. 5 (4.5 + E5) ~ 6 × 10 5 Cells were seeded at a density of one cell. The seeded cells were incubated at 37°C in an environment supplied with 5% CO2 and 5% O2.

[0192] 3.4.3 Trait introduction To infect iPS cells with a viral vector, the cells were grown to approximately 50-60% confluence (this can take 2-4 days without supporting cells). Once 50-60% confluence was reached, one well from the 12-well plate was isolated, and the total number of cells per well was determined by cell counting. The iPS cell wells were then infected with the AAV vector listed below at a predetermined MOI (see Tables 18 and 19). Before transduction, the old iPS cell medium was removed from the plate, and 1 ml of fresh iPS cell medium was added to each well. A predetermined volume of virus from the stock solution was added directly to each well. For information on the total number of infected cells, MOI, and volume of virus used for infection, please refer to Tables 18 and 19. Then, 5% CO2 Cells were incubated at 37°C for 18-48 hours in an environment supplied with 5% O2. Transduction was performed. After 18 - 24 hours of transduction, cells were observed microscopically for abnormalities or cell death. At this time point, an additional 1 ml of fresh iPS cell medium was added to each well containing infected and non - infected cells, and incubation was continued for an additional 18 - 24 hours at 37 °C in an environment supplied with 5% CO2 and 5% O2. Before harvesting, cells were observed microscopically to assess cell death or abnormal appearance.

[0193]

Table 20

[0194]

Table 21

[0195] Outcome measurement method - Western blot analysis was performed as described herein.

[0196] Results 5.1 Expression of AAV2 - hCHM V1, V2a, V2b, V3a, V3b in JB588 iPS cell line: Monoclonal human REP - 1 - specific antibody detected one ~ 75 - 80 kDa single hREP - 1 protein in transduced JB 588 iPS cells (data not shown). In the case of untreated control standards, no bands were observed, confirming the absence of the disease (data not shown). The intensity of the REP - 1 5 protein band at an MOI of 3×10 was higher in all vectors compared to an MOI of 1×10 5 and Strong efficacy was observed. Recombinant AAV2.hCHM virus-mediated delivery of the hCHM gene to iPS cells resulted in dose-dependent production of the REP-1 protein. Blot exploration using the GAPDH antibody showed bands of equal concentration in all cell lysates. GAPDH detected the protein at ~39 kDa. Both the REP-1 and GAPDH antibodies detected only specific bands at the predicted molecular weight. No nonspecific bands were observed in the blots.

[0197] Expression of AAV8-hCHM.V1 and V2a in iPS cells: Monoclonal human REP-1 specific antibody detected a single ~75-80 kDa mono-REP-1 protein in transduced iPS cells from subjects JB527, JB500, and JB588 (data not shown). No protein band was observed in the case of untreated control standards (data not shown). Blot exploration using GAPDH antibody showed bands of equal concentration in all cell lysates, including cell lysates from untreated cells. Anti-GAPDH antibody detected a specific ~39 kDa protein band for REP-1 and GAPDH. Both H antibodies detected only specific protein bands at the predicted size and molecular weight. No detectable nonspecific protein bands were observed in the blot.

[0198] conclusion The preliminary results presented in this report revealed the following observations: Western blot analysis confirmed the presence of CHM (deficiency of REP-1 protein) in each of three subject-derived iPSCs (JB588, JB500, JB527). In vitro expression studies showed that infection of iPS cells from CHM subjects with AAV2.hCHM. versions 2a, 2b, 3a, 3b and AAV2.hCHM version 1 (current clinical trial candidate) induced REP-1 protein production in all examined MOIs. 1 × 10⁶ of iPS cells were infected with AAV8.hCHM. versions 2a and AAV8.hCHM version 1. 6Infection of iPS cells at the MOI was observed in all three CHM iPS cell lines. This resulted in the production of the REP1 protein. The level of REP1 production was higher in iPSCs infected with AAV8.hCHM.V2a than in iPSCs infected with AAV8.hCHM.V1.

[0199] Example 7: Comparison of in vivo expression of AAV8 codon-optimized human CHM versus AAV, natural, and human CHM. Gene therapy for multiple retinal diseases relies on the effective transduction of appropriate target cells, and in the case of colloideremia, appropriate target cells are retinal pigment epithelium (RPE) and photoreceptor cells. This study report focuses on comparing in vivo expression in wild-type mice induced by a construct (version 1) based on the native CHM sequence and four next-generation transgene cassettes packaged in the AAV8 backbone. Here, we evaluated AAV8 serotypes for the purpose of improving gene transfer to photoreceptor cells.

[0200] Our experiment was designed to answer the following questions: a. How do these vectors compare in terms of in vivo transduction of photoreceptors? In particular, how effectively does next-generation AAV8.CHM transduce photoreceptors compared to version 1 after subretinal injection of each test substance? b. Dose-response: Do next-generation AAV8.CHM and the AAV8.CHM-version 1 vector differ in the dose-response of gene expression?

[0201] Experimental details: The plasmids and vectors were as described in Example 4. Mice (animals): Wild-type CD1 mice were used to investigate the in vivo expression of CHM, as assessed by the production of the REP-1 protein. The CD1 mouse strain is an uninbred Swiss mouse strain, and we maintain its colonies within the university. Details of the study are described under the CAROT research protocol PCPR02.01.

[0202] 3.3 Research Design (e.g., Processing Groups) 3.3.1 Animal Rearing: Both male and female mice (~3-4 months old) weighing ~20-30 grams were injected with the test substance described. The animals were housed in the University of Pennsylvania's John Morgan University Laboratory Animal Resources (ULAR) facility in accordance with the University of Pennsylvania's ULAR regulations. The mice were kept in a 12-hour light / 12-hour dark cycle. Food and water were provided as needed. All animals were identified by ear tag numbers.

[0203] 3.4 Test Material Administration: The test material preparation provided by CAROT Vector Core was used for dose administration. The test material was stored at -60 to -80°C. The test material was thawed on ice before administration. For intraocular injection, it was administered as described in Preparation Table 20. The test substance was diluted to the target concentration using phosphate-buffered saline. A total of 60 μl of master mix was prepared.

[0204] [Table 22]

[0205] Preparation of the injection log before subretinal injection. Prior to subretinal injection of the test substance, the injection log was kept along with the following information: • Gauge number / Mouse number Identification of the research ·KK ·date of birth ·Injection date • Name of the researcher / injector • The eye to be injected into (left or right) • Materials to be injected (vector / serotype) • Dosage and volume • Route of administration (ROA)

[0206] Subretinal injection: The injection was performed by a surgeon via subretinal injection. Briefly, the animals were anesthetized before the injection. A Hamilton 33G syringe was used to perform subretinal injection of the test substance. The details of the test substance and the injection are described in Table 21. From the prepared injection master mixture, a volume of 1.5 μl per injection was administered. One eye per animal received 5×10 8 individuals of vector genomes (vg), and the contralateral eye received 5×10 9 individuals of vector genomes were injected.

[0207]

Table 23

[0208] Outcome measurement method Animal Sacrifice: a. After injecting the test substance into the animals, all animals were observed for 48 hours for injection-related abnormalities. B. From 21 to 35 days after injection, the animals were observed for eye abnormalities using fundus examination methods. C. From 90 to 12 days after injection, the animals were sacrificed, and eye tissues were collected for evaluation of the production of exogenous REP-1 protein by SDS-PAGE and subsequent Western blot analysis.

[0209] Collection of eye tissues: After removing the lens from the eye using a sharp surgical scalpel blade, eye tissues for Western blot analysis were collected. The eyes (without the lens) were collected in freezer tubes and appropriately labeled.

[0210] Western blot analysis Briefly: 1. Preparation of tissue cell lysate a. Eye tissues of animals injected with two doses of next-generation AAV8.CHM and AAV8.V1 were collected 21 - 35 days after injection by sacrificing the animals, together with non-injected control standard animal tissues. B. The tissues were then lysed on ice using RIPA buffer with protease inhibitors. The tissue cell lysate was clarified by centrifugation at 13,000 rpm for 10 minutes.

[0211] 2. Quantification and preparation of proteins a. Protein quantification of cell lysates was performed using the ThermoFisher Micro BCATM Protein Assay Kit according to the manufacturer's instructions. b. Protein concentration was determined by obtaining the OD reading at 562 nm. c. To evaluate the in vivo expression of CHM, 20-40 μg of the measured protein was loaded onto 4-12% Bis-Tris gels.

[0212] 3. SDS-PAGE and Western blot The protein gel was transferred onto a nitrocellulose membrane, blocked in milk, and incubated with the primary antibody. Anti-human REP-1 2F1 antibody (2F1, 1:1000 dilution) and / or anti-GAPDH antibody (1:1000 dilution) were used as primary antibodies. After washing the blot, HRP-conjugated anti-mouse IgG antibody and / or anti-rabbit IgG antibody at a concentration of 1:5000 were used as secondary antibodies. The blot was chemiluminescent using ECL reagent according to the manufacturer's instructions.

[0213] 4. Control Standard a) Load control standard: Anti-GA to show equal loading of protein in each well of the gel PDH antibodies were used as the loading control standard. Anti-GAPDH antibodies detect proteins of ~39 kDa. B) Positive control standard: Lysate of COS-7 cells transduced with AAV2.V2a was used as the positive control standard. C) Negative control standard: Eye tissue from animals that were not injected was used as the negative control standard.

[0214] Determination of sample values Quantitative analysis of Western blot analysis using ImageJ software. In short, the concentration-metric evaluations presented in this report are first normalized to the endogenous expression level of GAPDH protein in the corresponding sample. Then, the expression level is normalized again to the mean REP-1 expression level of the uninjected control standard.

[0215] Details of the concentration-based evaluation and doubling calculations used to demonstrate REP-1 protein expression are presented in Tables 22 and 23.

[0216] Simple explanation: 1. In Tables 22 and 23, the second column shows the raw values ​​of REP-1 protein, and the third column shows the raw values ​​of GAPDH protein. 2. The GAPDH value of each sample is first normalized to the GAPDH value of Animal-1 in AAV8.V1, and this is shown in the fourth column of Table 22. 3. The values ​​for each sample are normalized to the GAPDH values ​​of Animal-2 in AAV8.V1, and these are shown in the 5th column of Table 22. 4. Next, the REP-1 value (second column) is normalized to either the GAPDH normalized to animal 1 (fourth column) or the GAPDH normalized to animal 2 (fifth column). These are shown in columns 6 and 7, respectively. 5. The normalized REP-1 values ​​are then converted to relative values ​​(fold change). 6. Normalize each REP-1 value to the REP-1 expression in either Animal 1 or Animal 2 of the group injected with AAV8.V1, and express it as a relative value (columns 8 and 9). 7. Column 10 shows the mean relative values ​​for REP-1 protein expression.

[0217] result Comparison of CHM expression using natural CHM AAV8.V1 versus codon-optimized CHM vectors: AAV8.V2a, V2b, V3a, and V3b: Two different doses of each AAV8 vector in wild-type CD1 mice: 5 × 10⁶ per eye 9 High doses of individual vector genomes and 5× per eye 10 8A low dose of each vector genome was injected. The following results are for high and low doses of AA Describe the levels of REP1 protein after injection with V8.V1, AAV8.V2a, and AAV8.V3a.

[0218] High dose (5×10 9 vector genomes per eye) of the viral vector was injected into the animals. Comparison of the expression of AAV8.V1 versus AAV8.V2a and AAV8.V3a (vectors with stuffers) after injection. Western blot analysis using human anti-REP-1 antibody detected ~75 - 80 kDa hREP-1 protein bands in both (low and high dose injected) eye tissues of each animal treated with either the next generation AAV8.V2a or V3a or the original AAV8 version 1. Very weak (minimal) bands were observed for both non-injected control standard mice. Bands of increased intensity were observed in tissues transduced with the next generation vectors (AAV8V.2a and AAV8.V3a) compared to tissues transduced with version 1. Anti-GAPDH antibody showed ~39 kDa bands of equal intensity in all lanes of the Western blot including non-injected control standards. Pre-stained protein markers were used for comparison of the molecular weights of the proteins in question. Density measurement (quantification of expression levels) of the blot using ImageJ software showed an increase in the production of REP-1 in animals injected with one of the next generation AAV8 high and low dose constructs (V2a or V3a). (See Table 22 for values.)

[0219]

Table 24

[0220]

Table 25

[0221] Low dose (5 x 10 per eye) 8 A viral vector (with individual vector genomes) is injected into the animal Comparison of AAV8.V1 versus AAV8.V2a and AAV8.V3a expression in organisms. 5 x 10 8 This dose provides next-generation AAV8.V2a, V3a and AAV8.version 1 Western blot analysis of injected animal eye tissue with human anti-REP-1 antibody detected a ~75-80 kDa hREP-1 protein band in injected mouse tissue. A weak (minimal) band of REP-1 was observed in the ocular tissue cell lysates of both uninjected control standard mice. Increased intensity bands were observed in the tissue cell lysates transduced using the next-generation vector compared to those transduced using version 1. Anti-GAPDH antibody detected a protein band of equal intensity at ~39 kDa in all tissue lysates. This data is comparable to that obtained with next-generation V2 via AAV8. a. CHM delivery produces robust levels of REP-1 protein compared to levels produced after injection of AAV8.V3a or AAV8.V1.

[0222] Evaluation of the blot concentration measurement method using ImageJ software (quantification of expression levels) further demonstrated increased REP-1 production in animals injected with the next-generation AAV8.CHM construct (particularly V2a) compared to version 1. See Table 23 for numerical data.

[0223] Expression of AAV8.V2b in CD1 mice We simultaneously evaluated the effect of lambda stuffer on AAV vector construction using the ongoing study and qPCR titer analysis. We performed all animal injections for in vivo expression studies as described in study protocol PCPR.02 and collected all samples. After the qPCR studies for the lambda stuffer elements (mentioned above) were completed, we decided to perform Western blotting experiments solely to examine the expression of stuffer-less AAV vectors such as AAV8.2b and AAV8.3b, and to exclude them from further analysis (such as comparison with version 1).

[0224] Human anti-REP-1 antibody: 5 × 10 9 AAV8 with a high-dose vector genome copy ~75-80 kDa proteins were detected in the eye tissue of CD-1 mice injected with .2b (Figure 12A). 5 × 10 8 Animals that received AAV8.2b in small doses were: A very weak protein band was observed at ~75-80 kDa (Figure 12A). Lysates of ocular tissue from uninjected control standard animals did not show the presence of REP-1 protein. Anti-GAPDH antibody detected ~39 kDa protein in all ocular tissue lysates, including those from uninjected control standards. This data allows for the determination of the minimum dose for AAV8.2b.

[0225] Expression of AAV8.V3b in CD1 mice We performed Western blot analysis using an anti-REP-1 antibody on the eye tissue of CD1 mice (2 mice per group) injected with AAV8.3b, revealing the presence of a ~75-80 kDa protein in one eye injected with a low dose and in both eyes injected with a high dose of AAV8.3b. REP-1 expression was not detected in the eye tissue of uninjected mice (Figure 12B). The level of REP-1 produced was dose-dependent in animals injected with AAV8.3b. 9 Injection using individual vector genomes is performed on eyes (5 × 10) with low doses injected. 8induced more REP-1 compared to the (individual vector genomes). Anti-GAPDH antibody detected a ~39 kDa protein in the eye tissue cell lysates of all injected and non-injected animals.

[0226] These results revealed the following observations: 1) Next-generation vectors AAV8, versions 2a, 2b, 3a, and 3b can effectively transduce eye tissue. 2) Expression of the transgene (codon-optimized CHM) was detectable for all of the next-generation vectors. 3) Expression of the transgene (codon-optimized CHM) was dose-dependent. 4) AAV8, version 2a and AAV8, version 2b induced increased production of REP-1 protein in the eye tissue of CD-1 mice compared to AAV8, version 1. 5) There was variation in the exact levels of transgene protein production among eyes injected with the same dose, reflecting variability in the surgical delivery method. However, the differences in levels were low (5×10 8 individuals) and large between low (5×10 9 individuals) and high (5×10 9 individual vector genomes) doses. 6) AAV8.CHM.V2a and AAV8.V3a produced much higher levels of REP-1 protein production compared to AAV8. V1 after in vivo administration of a high dose (5×10

[0227] All published documents cited in this specification, including provisional patent application No. 62 / 266,789, filed on December 14, 2015, are hereby incorporated by reference in their entirety. Similarly, the sequence numbers referred to in this specification and appearing in the accompanying sequence listing are hereby incorporated by reference into the content of this specification. Although the invention has been described in reference to specific embodiments, it will be understood that modifications can be made without departing from the spirit of the invention. Such modifications are intended to be included within the scope of the appended claims.

Sequence Listing Free-Text

[0228] Table 26-1

[0229] Table 26-2

[0230] Table 26-3

[0231] Table 26-4

[0232] Table 26-5

[0233] Table 26-6

[0234] Table 26-7

[0235] Table 26-8

[0236] Table 26-9

[0237] Table 26-10

[0238] Table 26-11

Claims

1. An expression cassette containing the nucleic acid sequence of sequence number 9 or 11.

2. An adeno-associated virus (AAV) vector comprising an AAV capsid and a nucleic acid including an AAV inverted terminal repeat (ITR) sequence, a sequence encoding human cyclic nucleotide-sensitive channel alpha-3 (CNGA3), and an expression regulatory sequence that directs the expression of CNGA3 in host cells, wherein the sequence encoding CNGA3 includes SEQ ID NO: 9 or SEQ ID NO:

11.

3. The AAV vector according to claim 2, wherein the sequence encoding the CNGA3 sequence encodes the protein sequence of sequence number 10.

4. The AAV vector according to claim 2 or 3, wherein the expression regulatory sequence includes a promoter.

5. The AAV vector according to claim 4, wherein the promoter is a rhodopsin promoter, a rhodopsin kinase promoter, or an ophthalmic cell-specific promoter.

6. The AAV vector according to claim 4, wherein the promoter is an induceable promoter, a constitutive promoter, or a tissue-specific promoter.

7. The AAV vector according to any one of claims 2 to 6, further comprising one or more introns, Kozak sequences, polyA, and post-transcriptional regulatory elements.

8. The AAV vector according to any one of claims 2 to 7, wherein the capsid is AAV2, AAV5, AAV8, AAV9, AAV8bp, or AAV7m8 capsid.

9. The AAV vector according to any one of claims 2 to 8, wherein the ITR sequence is derived from AAV2.

10. An AAV vector comprising an AAV8 capsid and an expression cassette, wherein the expression cassette comprises a nucleic acid sequence encoding CNGA3, an inverted terminal repeat (ITR) sequence, and an expression regulatory sequence that directs the expression of CNGA3 in host cells, the nucleic acid sequence encoding CNGA3 comprising SEQ ID NO: 9 or SEQ ID NO:

11.

11. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an AAV vector according to at least one of claims 2 to 10.

12. A plasmid for producing an AAV vector, comprising the expression cassette described in claim 1.

13. A composition comprising the AAV vector according to any one of claims 2 to 10 for the treatment of color blindness.

14. The composition according to claim 13, wherein the composition is administered subretin.

15. The composition according to claim 13, wherein the composition is administered intravitreously, intravenously, or choroidally.

16. The composition is about 10 9 pieces ~ about 10 13 The composition according to any one of claims 13 to 15, administered in a dose of an AAV vector containing a vector genome (VG).

17. The composition according to any one of claims 13 to 16, wherein the composition is administered in a volume of about 100 μL to about 500 μL.

18. Use of an AAV vector according to any one of claims 2 to 10 in the manufacture of a drug for treating color blindness.

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