Identification of mutations in channel opsin variants with improved photosensitivity and methods of use thereof

Abstract

The present invention provides compositions and kits comprising at least one nucleic acid or polypeptide molecule encoding a mutant CoChop protein. The methods of the invention comprise administering to a subject a composition comprising a mutant CoChop to protect, improve, or restore light transduction. Preferably, the compositions and methods of the invention are provided to a subject with visual impairment to restore vision to a normal level.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 U.S.C. 119(c) to U.S. Provisional Application Serial No. 62 / 380,871, filed on August 29, 2016, the contents of which are incorporated herein by reference in their entirety.

[0003] Government support

[0004] This invention was made with U.S. government support under National Institutes of Health / National Eye Institute Grant No. NIH EY 17130. The U.S. government has certain rights in this invention.

[0005] Description of the text file submitted electronically

[0006] The contents of the text files submitted electronically with this document are incorporated herein by reference in their entirety: Computer readable form copy of the Sequence Listing (file name: RTRO-707 / 001WO_SeqList_ST25.txt, recorded date, August 29, 2017), file size 24kb. Technical Field

[0007] The present invention relates to the field of molecular biology. Mutations in the channel opsin variant gene (CoChop) are identified. Compositions comprising mutant CoChop genes are used in therapeutic methods. For example, compositions comprising mutant CoChop genes improve and restore vision loss. Background of the Invention

[0009] The retina is composed of photoreceptors (or photoreceptor cells, rods and cones.) Photoreceptors are highly specialized neurons that are responsible for phototransduction, or the conversion of light (in the form of electromagnetic radiation) into electrical and chemical signals that propagate a chain of events within the visual system, ultimately generating our view of the world.

[0010] Photoreceptor loss or degeneration severely affects, if not completely inhibits, the phototransduction of visual information within the retina. Loss of photoreceptor cells and / or loss of photoreceptor cell function is a major cause of diminished visual acuity, diminished photosensitivity, and blindness. There is a long-felt need in the art for compositions and methods for restoring photosensitivity to the retina of a subject who has experienced vision loss. Summary of the invention

[0011] The present invention provides an isolated light-activated ion channel polypeptide having an amino acid sequence of SEQ ID NO: 2 and one or more amino acid modifications. The advantage of the CoChR mutants disclosed herein (e.g., mutant CoChop) is that these mutant polypeptides require less light than the wild-type CoChR (SEQ ID NO: 2) for activation. Therefore, at the same light intensity, greater ion flow and / or proton flow levels are observed in the mutant CoChR polypeptide than in the wild type. In some embodiments, when expressed in a cell membrane and contacted with an activating light, the light-activated ion channel polypeptide has at least one of the following: greater ion flow levels and greater proton flow levels (e.g., exceeding the activation threshold) compared to the light-activated ion channel polypeptide SEQ ID NO: 2. The light-activated ion channel polypeptide has an amino acid sequence of any one of SEQ ID NOs: 3-10. Optionally, the polypeptide further comprises one or more amino acid modifications, such as substitutions, deletions, or insertions.

[0012] In another aspect, the present invention provides an isolated nucleic acid molecule encoding a polypeptide of the present invention. Optionally, the nucleic acid sequence is operably linked to a promoter sequence. The present invention also includes a vector containing a nucleic acid according to the present invention.

[0013] The present invention also includes cells containing a polypeptide or nucleic acid according to the present invention. The cell is, for example, a photoreceptor, a bipolar cell, a rod bipolar cell, an ON-type cone bipolar cell, a retinal ganglion cell, a photosensitive retinal ganglion cell, a horizontal cell, an amacrine cell or an AII-type amacrine cell. The cell is in vitro, ex vivo or in vivo.

[0014] In other aspects, the present invention provides a method for changing the conductivity of a membrane by expressing a polypeptide of the present invention in the host membrane and contacting the polypeptide with light under suitable conditions to change the conductivity of the host membrane. The host membrane is a cell membrane, such as a neuronal cell, a nervous system cell, a cardiac muscle cell, a circulatory cell, a visual system cell, or an auditory system cell.

[0015] On the other hand, the invention provides methods for treating a disease or condition in a subject, comprising administering a therapeutically effective amount of a nucleic acid or polypeptide according to the invention to a subject in need thereof. The disease or condition is, for example, injury, brain injury, spinal cord injury, epilepsy, metabolic disorders, cardiac dysfunction, visual loss, blindness, deafness, hearing loss, or a neurological condition.

[0016] In another aspect, the invention relates to a method for improving or restoring vision by administering a nucleic acid or polypeptide according to the invention to a subject suffering from an eye disease such as macular degeneration or retinitis pigmentosa.

[0017] Improving or restoring vision includes, for example, increasing light sensitivity; lowering the threshold light intensity required to elicit photocurrents; increasing visual evoked potentials in the visual cortex; and lowering the threshold light intensity to elicit visually guided behaviors, such as optokinetic responses.

[0018] In another aspect, the present invention provides methods for treating retinitis pigmentosa or age-related macular degeneration, which methods comprise administering a nucleic acid or polypeptide according to the present invention to a subject in need thereof. The composition is administered by intravitreal or subretinal injection.

[0019] Other features and advantages of the invention will be apparent from and encompassed by the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Comparison of spectral curves of CoChR and ChR2 in HEK cell recordings. The peak spectrum of CoChR is ~480nm, which is slightly more red-shifted than the spectrum of ChR2.

[0021] Figure 2 : Sample recordings of light-evoked currents of CoChR and ChR2 in HEK cell recordings. AD are light-evoked currents of wt-ChR2 (A) and its three mutants ChR2-L132C (B), ChR2-L132C / T159C (C), and ChR2-L132C / T159C (D). Currents were evoked by increasing light intensities using a neutral density (ND) filter at ND=0, 2.5, 3.0, and 4.0. The red trace was generated by 460 nm light at 2.5 neutral density (ND) (4.1 x 10 15 Photons / cm 2 s). E and F are light-evoked currents of wt-CoChR (E) and its mutant CoChR-L112C (F). The red trace was induced by 480 nm light at 2.5 neutral density (ND) (4.8x10 15 Photons / cm 2 s) triggered.

[0022] Figure 3: Comparison of current amplitudes of wt-CoChR and its more light-sensitive mutants CoChR-L112C (SEQ ID NO: 3), CoChR-T139C (SEQ ID NO: 5), CoChR-L112C / T139C (SEQ ID NO: 8), CoChR-T145A / S146A (SEQ ID NO: 6), CoChR-L112C / H94E (SEQ ID NO: 9), and CoChR-L112C / H94E / K264T (SEQ ID NO: 10) in HEK cell recordings. The current was induced by 480 nm light at ND=2.5 (4.8×10 15 Photons / cm 2 s) induced and normalized to wt-CoChR. Data are shown as mean ± SD.

[0023] Figure 4 : Comparison of decay time constants (dissociation rates) of wt-CoChR and its more light-sensitive mutants CoChR-L112C, CoChR-T139C, CoChR-L112C / T139C, CoChR-T145A / S146A, CoChR-L112C / H94E, and CoChR-L112C / H94E / K264T in HEK cell recordings. 18 Photons / cm 2 s) Evoked current. Data are shown as mean ± SD.

[0024] Figure 5 : Relationship between the amplitude of light-evoked current and the decay time constant (or dissociation rate) of wt-CoChR and its more light-sensitive mutants CoChR-L112C, CoChR-L112C / T139C, CoChR-L112C / H94E and CoChR-L112C / H94E / K264T in HEK cell recordings.

[0025] Figure 6 : Comparison of the light sensitivity of ChR2-L132C / T159C, wt-CoChR, and CoChR-L112C in retinal ganglion cells with multielectrode array recordings. Light intensity was expressed as neutral density (ND) and photons / cm 2 s shown.

[0026] Figure 7: Optokinetic behavioral test for comparing the light sensitivity of restored optokinetic responses between ChR2-L132C / T159S and CoChR-L112C viral vector injected mice. Relationship between the spectral frequency required to induce optokinetic responses of ChR2-L132C / T159S and CoChR-L112C and the threshold light intensity. Experiments were performed using a blind mouse strain. Optokinetic tests were performed in a domestic optokinetic assay system. Light stimulation was generated by a blue LED with a wavelength of ~470nm. The threshold light intensity for inducing optokinetic responses in CoChR-L112C expressing mice was approximately 2-3x10 13 Photons / cm 2 s, and the threshold light intensity for eliciting optokinetic responses in ChR2-L132C / T159S-expressing mice was approximately 1-2x10 14 Photons / cm 2 s. Data are shown as mean ± SD.

[0027] Figure 8 : Contrast sensitivity curves of CoChR-L112C viral vector-injected mice based on optokinetic behavioral testing. Experiments were performed using a blind mouse strain. Optomotor testing was performed in an optokinetic system (OptoMotry; Cerebral Mechanics, Lethbridge, AB, Canada). The illumination within the platform was ~150 lux. Data are shown as mean ± SD.

[0028] Figure 9: Long-term stable expression of wt-CoChR and CoChR-L112C in retinal neurons mediated by AAV vector delivery. A and B, fluorescent images show the expression of wt-CoChR and its mutant CoChR-L112C in retinal ganglion cells of C57BL / 6J mice one month after viral injection. C and D, fluorescent images show the expression of wt-CoChR and its mutant CoChR-L112C in retinal ganglion cells of rd1 mice six months after viral injection. EG, fluorescent images show the expression of CoChR-L112C in the retina of a blind mouse strain nine months after viral injection, viewed in whole-mount at low resolution (E) and high resolution (F) and viewed in vertical sections of the retina (G). DETAILED DESCRIPTION

[0029] The present invention is based in part on the unexpected discovery that mutations in channel opsin variants from the green algae, Chloromonas oogama, CoChop result in increased light sensitivity. The CoChop mutant amino acid and nucleic acid sequences according to the present invention are referred to herein as mCoChop. Wild-type CoChop is described, for example, in WO2015 / 161308, the contents of which are incorporated by reference as a whole. The mCoChop amino acid and nucleic acid sequences according to the present invention are suitable for any application in which an activated ion channel is desired.

[0030] In a specific embodiment, the present invention relates to compositions and methods for treating retinal degenerative diseases, such as retinitis pigmentosa or age-related macular degeneration. In addition, other diseases and conditions that are a direct result of retinal degenerative diseases are also treated by the methods of the present invention. For example, advanced retinitis pigmentosa and other retinal degenerative conditions lead to macular degeneration.

[0031] The channel opsin variant CoChop was first identified by resequencing the 127 algal transcriptome. CoChop was identified by synthesis and screening of photocurrents in HEK293 cells. (See WO2015 / 161308 and Klapoetke et al. Nature Methods Vol. 11, No. 3 2014, the contents of each of which are incorporated by reference in their entirety.)

[0032] As referred to herein, "CoChop" refers to a gene encoding a channel opsin that forms a channel rhodopsin (CoChR) once bound to the retina. The genetic constructs of the present invention primarily refer to CoChop (i.e., without retinal), and all CoChop mutants (mCoChop) disclosed herein form functional channel rhodopsin (ChR). The methods disclosed herein may include delivering mCoChop to cells with or without exogenous retinal. It should be understood that when mCoChop is expressed in cells (i.e., retinal neurons), endogenous available retinal binds to the mCoChop protein of the present invention to form a functional light-gated channel. In this way, the Chop protein as referred to herein may also be synonymous with ChR.

[0033] The following sequences provide non-limiting examples of wild-type CoChop mutant CoChop proteins and polynucleotides encoding said WT and mutant Chop proteins of the present invention and forming the WT and mutant ChRs of the present invention.

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045] The present invention also encompasses CoChop proteins and nucleic acids encoding biologically active fragments of CoChop or variants of conservative amino acid substitutions or other mutations. Smaller fragments of wild-type CoChop may also be suitable for use in the present invention, in which at least one amino acid is mutated or conservatively substituted. In other embodiments, the CoChop polypeptides and nucleic acids of the present invention may be up to or about 275 amino acids in length, 250 amino acids in length, 225 amino acids in length, 200 amino acids in length, 175 amino acids in length, or 160 amino acids in length.

[0046] In some embodiments, the present disclosure provides derivatives, variants or mutants of one or more CoChop polypeptides disclosed herein. In some embodiments, the derivative, variant or mutant contains one or more amino acid substitutions compared to the amino acid sequence of a natural polypeptide (e.g., SEQ ID NO: 2). In some embodiments, one to 20 amino acids are substituted. In some embodiments, the derivative, variant or mutant contains about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 amino acid substitutions compared to the amino acid sequence of a natural therapeutic peptide agent. In some embodiments, the derivative, variant or mutant contains one or more amino acid deletions compared to the amino acid sequence of a natural polypeptide (e.g., SEQ ID NO: 2). In some embodiments, one to 20 amino acids are deleted compared to the amino acid sequence of a natural polypeptide (e.g., SEQ ID NO: 2). In some embodiments, the derivative, variant or mutant has about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 amino acid deletions compared to the amino acid sequence of a natural polypeptide (e.g., SEQ ID NO: 2). In some embodiments, one to ten amino acids are deleted at either end compared to the amino acid sequence of a native polypeptide (e.g., SEQ ID NO: 2). In some embodiments, one to ten amino acids are deleted at both ends compared to the amino acid sequence of a native polypeptide (e.g., SEQ ID NO: 2). In some embodiments, the amino acid sequence of the derivative, variant, or mutant is at least about 70% identical to the amino acid sequence of a native polypeptide (e.g., SEQ ID NO: 2). In some embodiments, the amino acid sequence of the derivative, variant, or mutant is about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence of a native polypeptide (e.g., SEQ ID NO: 2).

[0047] The mutant CoChop proteins of the present invention also demonstrate slower channel dynamics. It was found that higher light sensitivity was associated with lower channel dynamics, which indicates a trade-off between light sensitivity and channel dynamics. The mCoChop proteins that can form the ChR proteins of the present invention can also include additional mutations or modifications that can improve channel dynamics or increase the inactivation rate. Particularly preferred CoChop mutants balance the threshold of light sensitivity with channel dynamics.

[0048] For example, the mutant ChR protein of the present invention achieves greater light sensitivity by extending the channel open state. Therefore, each mutant ChR channel conducts a larger photocurrent than the wild-type ChR channel when activated by the same light intensity. Therefore, the mutant channel is activated by light intensities lower than those required for the activation of the wild-type ChR channel. Quantitatively, the detectable peak activity of retinal ganglion cells expressing mutant ChR proteins can be triggered by light intensity, which is 1.5-2log units lower than the light intensity required for retinal ganglion cells expressing wild-type ChR to trigger peak activity. Therefore, the light intensity required to activate the mutant ChR protein is close to or falls under normal outdoor lighting conditions.

[0049] Nucleic acids, vectors and recombinant viruses

[0050] In some aspects of the invention, the compositions and methods of the present disclosure provide for delivery of cells encoding mCoChop (mutant CoChop) to a subject or patient in need thereof. In some cases, delivery of nucleic acids may be referred to as gene therapy.

[0051] The compositions and methods disclosed herein provide any suitable method for delivering mCoChop nucleic acids. In some cases, the delivery of nucleic acids can be performed using any suitable "carrier" (sometimes also referred to as "gene delivery" or "gene transfer" vector). Carriers, delivery vehicles, gene delivery vehicles or gene transfer vehicles may refer to any suitable macromolecules or a complex of molecules comprising polynucleotides delivered to target cells. In some cases, the target cell can be any cell that delivers nucleic acids or genes. The polynucleotides to be delivered can include a coding sequence of interest in gene therapy, such as an mCoChop gene.

[0052] For example, suitable vectors may include, but are not limited to, viral vectors such as adenovirus, adeno-associated virus (AAV), and retrovirus, liposomes, other lipid-containing complexes, and other macromolecular complexes capable of mediating the delivery of a polynucleotide to a target cell.

[0053] In some cases, the carrier can be an organic or inorganic molecule. In some cases, the carrier can be a small molecule (ie, <5 kD) or a macromolecule (ie, >5 kD). For example, the carrier can include, but is not limited to, an inert non-biologically active molecule, such as a metal particle. In some cases, the carrier can be a gold particle.

[0054] In some aspects, the vector may include a recombinant viral vector in combination with one or more nucleic acids. As described herein, nucleic acid may refer to a polynucleotide. Nucleic acid and polynucleotide can be used interchangeably. In some cases, nucleic acid may include DNA or RNA. In some aspects, nucleic acid may include DNA or RNA for expressing mCoChop. In some aspects, RNA nucleic acid may include but is not limited to transcripts (e.g., mCoChop), introns, untranslated regions, termination sequences, etc. of genes of interest. In other cases, DNA nucleic acid may include but is not limited to the following sequences, such as hybrid promoter gene sequences, strong constitutive promoter sequences, genes of interest (e.g., mCoChop), untranslated regions, termination sequences, etc. In some cases, a combination of DNA and RNA may be used.

[0055] As described in the disclosure herein, the term "expression construct" means any type of genetic construct comprising a nucleic acid or polynucleotide containing a coding gene product, wherein a portion or all of the nucleic acid coding sequence can be transcribed. The transcript can be translated into a protein. In some aspects, it can be partially translated or not translated. In some aspects, expression includes transcription of a gene and translation of mRNA to a gene product. In other aspects, expression only includes transcription of a nucleic acid encoding a gene of interest.

[0056] In one aspect, the present disclosure provides a recombinant virus, such as adeno-associated virus (rAAV) as a vector to mediate the expression of mCoChop.

[0057] In some cases, the viral vectors of the present disclosure may be measured as pfu (plaque forming units). In some cases, the pfu of the recombinant virus or viral vector of the compositions and methods of the present disclosure may be about 10 8 About 5x10 10 In some cases, the recombinant virus of the present disclosure is at least about 1×10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 6x10 8 , 7x10 8 , 8x10 8 , 9x10 8 , 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 , 9x10 9 , 1x1010 , 2x10 10 , 3x10 10 , 4x10 10 , and 5x10 10 In some cases, the recombinant virus of the present disclosure is at most about 1×10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 6x10 8 , 7x10 8 , 8x10 8 , 9x10 8 , 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 , 9x10 9 , 1x10 10 , 2x10 10 , 3x10 10 , 4x10 10 , and 5x10 10 pfu.

[0058] In some cases, the viral vectors of the present disclosure can be measured as vector genomes. In some cases, the recombinant viruses of the present disclosure are 1x10 10 Up to 3x10 12 In some cases, the recombinant virus of the present disclosure is 1×10 9 Up to 3x10 13 In some cases, the recombinant virus of the present disclosure is 1×10 8 Up to 3x10 14 In some cases, the recombinant virus of the present disclosure is at least about 1×10 1 , 1x10 2 , 1x10 3 , 1x10 4 , 1x10 5 , 1x10 6 , 1x10 7 , 1x10 8 , 1x10 9 , 1x10 10 , 1x10 11 , 1x10 12 , 1x1013 , 1x10 14 , 1x10 15 , 1x10 16 , 1x10 17 , and 1x10 18 vector genome.

[0059] In some cases, the viral vectors of the present disclosure can be measured as a multiplicity of infection (MOI). In some cases, MOI can refer to the ratio or multiplicity of vector or viral genome to cells to which nucleic acid can be delivered. In some cases, MOI can be 1x10 6 In some cases, the MOI can be 1x10 5 -1x10 7 In some cases, the MOI can be 1x10 4 -1x10 8 In some cases, the recombinant virus of the present disclosure is at least about 1×10 1 , 1x10 2 , 1x10 3 , 1x10 4 , 1x10 5 , 1x10 6 , 1x10 7 , 1x10 8 , 1x10 9 , 1x10 10 , 1x10 11 , 1x10 12 , 1x10 13 , 1x10 14 , 1x10 15 , 1x10 16 , 1x10 17 , and 1x10 18 In some cases, the MOI of the recombinant virus of the present disclosure is 1×10 8 Up to 3x10 14 MOI.

[0060] In some aspects, nucleic acid can be delivered without the use of a virus (i.e., using a non-viral vector) and can be measured as the amount of nucleic acid. Generally, any suitable amount of nucleic acid can be used in the compositions and methods of the present disclosure. In some cases, the nucleic acid can be at least about 1 pg, 10 pg, 100 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 μg, 10 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 pg, 1 μg, 10 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μ ... g, 900μg, 1ng, 10ng, 100ng, 200ng, 300ng, 400ng, 500ng, 600ng, 700ng, 800ng, 900ng, 1mg , 10mg, 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg1g, 2g, 3g, 4g, or 5g. In some cases, the nucleic acid may be at most about 1 pg, 10 pg, 100 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 μg, 10 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, In some aspects, a self-complementary vector (sc) can be used. The use of a self-complementary AAV vector can bypass the need for viral second strand DNA synthesis and can result in greater transgenic protein expression rates, as provided by Wu, Hum Gene Ther. 2007, 18(2): 171-82, which is incorporated herein by reference.

[0061] The compositions and methods of the present disclosure provide any suitable viral nucleic acid delivery system, including but not limited to the use of at least one of adeno-associated virus (AAV), adenovirus, helper-dependent adenovirus, retrovirus, herpes simplex virus, lentivirus, poxvirus, hemagglutinating virus of Japanese liposome (HVJ) complex, Moloney murine leukemia virus, and HIV-based viruses. Preferably, the viral vector comprises a strong eukaryotic promoter operably linked to the polynucleotide.

[0062] Generally, any suitable viral vector can be engineered to optimize the compositions and methods of the present disclosure. For example, a viral vector derived from adenovirus (Ad) or adeno-associated virus (AAV) can be used. Human and non-human viral vectors can be used and the recombinant viral vector can be changed so that it may be replication-defective in humans. When the vector is an adenovirus, the vector may include a polynucleotide having a promoter operably linked to a gene encoding the mCoChop protein and is replication-defective in humans.

[0063] In order to combine the advantageous properties of the two viral vector systems, hybrid viral vectors can be used to deliver nucleic acids encoding mCoChop proteins to target cells or tissues. Standard techniques for constructing hybrid vectors are well known to those skilled in the art. Such techniques can be found, for example, in Sambrook et al., In Molecular Cloning: A laboratory manual. Cold Spring Harbor, NY, or any number of laboratory manuals discussing recombinant DNA technology. Double-stranded AAV genomes in adenoviral capsids containing a combination of AAV and adenoviral ITRs can be used to transduce cells. In another variation, the AAV vector can be placed in a "viral gene-free," "helper-dependent," or "high-capacity" adenoviral vector. Adenovirus / AAV hybrid vectors are discussed in Lieber et al., J. Virol. 73: 9314-9324, 1999. Retrovirus / adenovirus hybrid vectors are discussed in Zheng et al., Nature Biotechnol. 18: 176-186, 2000.

[0064] The retroviral genome contained in the adenovirus can be integrated into the target cell genome and achieve stable gene expression.

[0065] Replication-deficient recombinant adenoviral vectors can be generated according to known techniques, see Quantin et al., Proc. Natl. Acad. Sci. USA, 89: 2581-2584 (1992); Stratford-Perricadet et al., J. Clin. Invest., 90: 626-630 (1992) and Rosenfeld et al., Cell, 68: 143-155 (1992).

[0066] In addition, preferred vectors may include, but are not limited to, viral vectors, fusion proteins, and chemical conjugates. Retroviral vectors include Moloney murine leukemia virus and HIV-based viruses. In some cases, HIV-based viral vectors may be used, wherein the HIV-based viral vector comprises at least two vectors, wherein the gag and pol genes are from the HIV genome and the env gene is from another virus. DNA viral vectors may be used. These vectors include poxvirus vectors, such as smallpox or fowlpox virus vectors, herpesvirus vectors, such as herpes simplex I virus (HSV) vectors [Geller, AI et al., J. Neurochem, 64:487 (1995); Lim, F. et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, AI et al., Proc Natl. Acad. Sci.: USA: 907603 (1993); Geller, AI et al., Proc Natl. Acad. Sci. USA: 87:1149 (1990)], adenovirus vectors [LeGal ... USA: 87:1149 (1990)], LaSalle et al., Science, 259:988 (1993); Davidson et al., Nat. Genet. 3:219 (1993); Yang et al., J. Virol. 69:2004 (1995)], and adeno-associated virus vectors [Kaplitt, MG et al., Nat. Genet. 8:148 (1994)], which are incorporated herein by reference.

[0067] Other viral vectors that can be used according to the present disclosure include vectors based on herpes simplex virus (HSV). HSV vectors that lack one or more immediate early genes (IE) are advantageous because they are generally non-cytotoxic, persist in a state similar to latency in target cells, and provide efficient target cell transduction. Recombinant HSV vectors can incorporate approximately 30 kb of heterologous nucleic acid.

[0068] Retroviruses, such as type C retroviruses and lentiviruses, may also be used in the present disclosure. For example, a retroviral vector may be based on murine leukemia virus (MLV), as provided by Hu and Pathak, Pharmacol. Rev. 52: 493511, 2000 and Fong et al., Crit. Rev. Ther. Drug Carrier Syst. 17: 1-60, 2000, which are incorporated herein by reference. MLV-based vectors may contain up to 8 kb of heterologous (therapeutic) DNA in place of viral genes. Additional retroviral vectors may be used, including but not limited to vectors based on replication-defective lentiviruses, including vectors based on human immunodeficiency virus (HIV), as provided by Vigna and Naldini, J. Gene Med. 5: 308-316, 2000 and Miyoshi et al., J. Virol. 72: 8150-8157, 1998, which are incorporated herein by reference. Lentiviral vectors may be advantageous in that they are able to infect both actively dividing and non-dividing cells. They may also be highly efficient at transducing human epithelial cells.

[0069] The lentiviral vector used in the present disclosure can be derived from human and non-human (including SIV) lentivirus. Examples of lentiviral vectors include nucleic acid sequences required for vector propagation and tissue-specific promoters that can be operably linked to the mCoChop gene. The nucleic acid sequence may include viral LTR, primer binding sites, polypurine sequences, att sites, and encapsidation sites.

[0070] Lentiviral vectors can be encapsulated into any suitable lentiviral capsid. Replacing one particle protein with another protein from a different virus is called "pseudotyping". Vector capsids can contain viral envelope proteins from other viruses, including murine leukemia virus (MLV) or vesicular stomatitis virus (VSV). Use of the VSV G-protein produces high vector titers and greater stability of vector virus particles.

[0071] Alphavirus-based vectors, such as those made from Semliki Forest Virus (SFV) and Sindbis Virus (SIN), can also be used in the present disclosure. The use of alphaviruses is described in Lundstrom, K., Intervirology 43: 247-257, 2000 and Perri et al., Journal of Virology 74: 9802-9807, 2000, which are incorporated herein by reference.

[0072] Recombinant replication-deficient alphavirus vectors can be advantageous because they are capable of high levels of heterologous (therapeutic) gene expression and can infect a wide range of target genes. Alphavirus replicons can be targeted to specific cell types by displaying on their virion surfaces functional heterologous ligands or binding domains that will allow selective binding to target cells expressing the cognate binding partner. Alphavirus replicons can have a latent period and thus carry out long-term heterologous nucleic acid expression in target cells. Replicons can also exhibit transient heterologous nucleic acid expression in target cells.

[0073] Poxvirus vectors can introduce genes into the cytoplasm. Fowlpoxvirus vectors can only produce short-term expression of genes or nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors can be used in the compositions and methods of the present disclosure. In some aspects, adenovirus vectors can produce shorter-term expression (e.g., less than about one month) than adeno-associated viruses, and can exhibit longer-term expression. The specific vector selected can depend on the target cell and the condition being treated.

[0074] Adeno-associated viruses (AAV) are small, non-enveloped, single-stranded DNA viruses. They are non-pathogenic human parvoviruses and can rely on helper viruses for replication, including adenovirus, herpes simplex virus, vaccinia virus, and CMV. Exposure to wild-type (wt) AAV has not been associated with or known to cause any human pathology and is common in the general population, usually occurring before the age of ten, associated with adenovirus infection.

[0075] As used herein, "AAV" refers to adeno-associated virus and "rAAV" refers to recombinant adeno-associated virus.

[0076] In some cases, wild-type AAV encodes rep and cap genes. The rep gene is required for viral replication and the cap gene is required for the synthesis of capsid proteins. Through the combination of alternative translation start sites and splice sites, the minigenome may be able to express four rep gene products and three cap gene products. The rep gene products and sequences in the terminal inverted repeats (145bp ITR, which flank the genome) may be crucial in this process. To date, 11 serotypes of AAV have been isolated. The compositions and methods of the present disclosure provide for the use of any suitable AAV serotype. In some aspects, AAV is selected from the group consisting of AAV1, AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10 and hybrids thereof. AAV2 can be used in the compositions and methods of the present disclosure.

[0077] AAV2 is the most characterized. rAAV2 has been shown to be able to mediate long-term transgene expression in the eyes of many animal species. In rats, rAAV-mediated reporter gene (green fluorescent protein) is still present 18 months after injection. In monkeys, the same reporter gene is present 17 months after injection.

[0078] The vector may include components or functions that further regulate gene delivery and / or gene expression or provide favorable properties to the target gene in addition. Such other components include, for example, components that affect binding or targeting to cells (including components that mediate cell type or tissue-specific binding); components that affect the uptake of vector nucleic acid by cells; components that affect the localization of polynucleotides in cells after uptake (such as agents that mediate nuclear localization); and components that affect the expression of polynucleotides. Such components may also include markers that can be used to detect or select cells that have absorbed and are expressing nucleic acids delivered by the vector, such as detectable and / or selectable markers. Such components can be provided as natural properties of the vector (such as using certain viral vectors with components or functions that mediate binding and uptake), or the vector may be modified to provide such functions.

[0079] Selectable markers can be positive, negative or bifunctional. Positive selectable markers allow the selection of cells carrying the marker, while negative selectable markers allow the selective removal of cells carrying the marker. A variety of such marker genes have been described, including bifunctional (i.e., positive / negative) markers (see, for example, Lupton, S., WO 92 / 08796, published on May 29, 1992; and Lupton, S., WO 94 / 28143, published on December 8, 1994). Examples of negative selectable markers may include resistance genes comprising antibiotics (such as ampicillin or kanamycin). Such marker genes can provide increased control measurements, which may be advantageous in the context of gene therapy. A large number of such vectors are known in the art and are generally available.

[0080] In many viral vectors compatible with the methods of the present disclosure, one or more promoters may be included in the vector to allow more than one heterologous gene to be expressed by the vector. In addition, the vector may contain sequences encoding signal peptides or other moieties that promote expression of mCoChop protein from target cells.

[0081] The nucleic acid encoding the gene product can be under the transcriptional control of the promoter. As provided herein, "promoter" refers to the suitable DNA sequence required for starting gene transcription. The phrase "under transcriptional control" means that the promoter is in the correct position and orientation relative to the nucleic acid to control RNA polymer initiation and gene expression. In some cases, the promoter may include a "strong" or constitutively active promoter. For example, a CMV promoter, such as a constitutively active promoter known in the art, may be used. In some cases, the CMV promoter may include additional regulatory elements for promoting expression.

[0082] In some cases, a promoter may refer to a "weak" promoter or a sequence that produces a lower level of mCoChop protein than a strong promoter. In some cases, a promoter may be used such that the promoter drives the selective expression of mCoChop. In some cases, a promoter or other regulatory element used in combination with other sequences as described herein may be used to drive the selective expression of mCoChop in eye cells or eye tissues.

[0083] In addition, "promoter" can also be used interchangeably in this article, referring to any additional suitable transcription control module that can exist around the start site of RNA polymerase. The compositions and methods of the present disclosure can use any suitable promoter and transcription control module for expressing transgenic. Additional transcription control modules may include but are not limited to elements such as HSV thymidine kinase (tk) and SV40 early transcription units. Generally, promoters can be composed of discrete functional modules, each of which is composed of about 7-20bp of DNA or 20-5000bp of DNA, and promoters contain one or more recognition sites of transcription activators or repressor proteins. The compositions and methods of the present disclosure provide any suitable regulatory sequence or combination thereof. In some cases, these transcription control module sequences can be referred to as or identified as enhancer or repressor sequences.

[0084] At least one module in each promoter is used to locate the synthetic start site of RNA. An example is the TATA box. Other examples may include some promoters lacking the TATA box, such as the promoter for mammalian terminal deoxynucleotidyl transferase gene and the promoter for SV40 late gene, and the discrete elements superimposed on the start site themselves help to fix the start position.

[0085] Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located in the region 30-110bp upstream of the start site, although many promoters may also contain functional elements downstream of the start site. The spacing between promoter elements can often be flexible, so that promoter function is retained when the elements are inverted or moved relative to each other. For example, in the tk promoter, the spacing between promoter elements can be increased to 50bp intervals before activity begins to decline. Depending on the promoter, individual elements can be positioned to operate together or act independently to initiate transcription.

[0086] The compositions and methods of the present disclosure provide any suitable sequence for controlling the expression of a nucleic acid sequence of interest in a target cell. Therefore, when targeting human cells, the nucleic acid coding region can be engineered to be adjacent to and under the control of a promoter that can be expressed in human cells. Typically, this promoter can include a human or viral promoter.

[0087] In various aspects of the present disclosure, the human cytomegalovirus (CMV) immediate early (IE) enhancer, chicken β-actin promoter, chicken β-actin exon 1, hybrid chicken β-actin and rabbit β-globin intron, simian virus 40 polyadenylation signal can be used to obtain high level expression of a coding sequence of interest (e.g., mCoChop).

[0088] Also contemplate the use of other viral or mammalian cell or bacterial phage promoters that are well known in the art to realize the expression of an interested coding sequence, as long as the expression level is sufficient for a given purpose. In some aspects, prokaryotic regulatory sequences may be present in the vector, such as the T7 RNA polymerase promoter sequence. In other aspects, the vector does not contain such regulatory sequences. By adopting a promoter with known properties, the level and pattern of protein expression can be optimized after transfection or conversion.

[0089] The selection of promoters that are regulated in response to specific physiological or synthetic signals can allow for inducible expression of gene products. For example, in the case where the expression of one or more transgenics is toxic to the cells in which the vector is produced when using a polycistronic vector, it may be desirable to suppress or reduce the expression of one or more transgenics. Examples of transgenics that may be toxic to production cell lines are pro-apoptotic and cytokine genes. Several inducible promoter systems can be used to produce viral vectors that may be toxic to transgenic products. The compositions and methods of the present disclosure provide any suitable combination of promoter sequences, regulatory sequences, and transgenics. In some cases, the combination of sequences may not be toxic to cells. In some cases, the combination of sequences may be highly toxic to cells. In some cases, the combination of sequences may produce moderate levels of toxicity to cells.

[0090] In some cases, it may be desirable to regulate the expression of the transgenic in the gene therapy vector. For example, different viral promoters with different activity strengths may be utilized, depending on the desired expression level. In mammalian cells, the CMV immediate early promoter may be used to provide strong transcriptional activation. When it is desired that the expression level of the transgenic be reduced, a modified version of the less efficient CMV promoter has also been used. When it is desired that the transgenic be expressed in hematopoietic cells, a retroviral promoter is usually used, such as using the LTR (long terminal repeat) from MLV or MMTV. Other viral promoters that may be used according to the desired effect include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenovirus promoters (such as from E1A, E2A or MLP regions), AAV LTR, cauliflower mosaic virus, HSV-TK and avian sarcoma virus.

[0091] In some cases, promoters or regulatory sequence elements can be used to direct selective expression in eye cells or eye tissues. For example, promoters, sequence elements or regulatory sequences found in specific eye cell types such as retinal pigment epithelial cells can be used in suitable expression constructs (e.g., RPE65 or VMD2 promoters).

[0092] The selection of an appropriate promoter can be readily accomplished. In some cases, a high expressing or strong promoter can be used.

[0093] Other elements that can enhance expression may also be included, such as enhancers or systems that lead to high levels of expression, such as tat genes and tar elements. This box can then be inserted into a vector containing, for example, an E. coli origin of replication, such as a plasmid vector, such as pUC19, pUC118, pBR322, or other known plasmid vectors. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory press, (1989). Promoters are discussed below. The plasmid vector may also contain a selectable marker, such as a beta-lactamase gene for ampicillin resistance, as long as the marker polypeptide does not adversely affect the metabolism of the organism being treated. The box may also be incorporated into a nucleic acid binding portion in a synthetic delivery system, such as the system disclosed in WO 95 / 22618, which is incorporated herein by reference. Typically, the promoter sequence and / or any associated regulatory sequences may comprise about at least 150bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp, 2000bp, 3000bp, 4000bp, 5000bp or 10000bp. The promoter sequence and any associated regulatory sequences may comprise about at most 150bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp, 2000bp, 3000bp, 4000bp, 5000bp or 10000bp.

[0094] In some aspects, the recombinant vector or plasmid comprises a promoter selected from the group consisting of a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter, and an MMT promoter, an EF-1α promoter, an UB6 promoter, a chicken β-actin promoter, a CAG promoter, an RPE65 promoter, and an opsin promoter.

[0095] In some aspects, antibiotic markers are used in the process for producing recombinant viruses. Antibiotic resistance markers can be used to identify positive transgenic cells in the generation of recombinant viruses. For example, the marker that confers resistance can include but is not limited to kanamycin, gentamicin, ampicillin, chloramphenicol, tetracycline, doxycycline or hygromycin. In some aspects, the antibiotic resistance gene is a non-β-lactam antibiotic resistance gene, such as kanamycin.

[0096] In some aspects, the recombinant virus and / or the plasmid used to generate the recombinant virus comprises a sequence encoding an origin of replication sequence, such as those provided herein. The origin of replication sequence generally provides a sequence suitable for propagating the plasmid.

[0097] In some aspects, the recombinant virus and / or the plasmid used to generate the recombinant virus comprises an enhancer, such as those provided herein. Preferably, the enhancer is the CMV immediate early enhancer.

[0098] In some aspects, the recombinant virus and / or the plasmid for generating the recombinant virus comprises a poly A (polyadenylation) sequence, such as those provided herein (e.g., SV40 poly A sequence). Generally, any suitable poly A sequence can be used for the desired expression of transgenic (i.e., mCoChop). For example, in some cases, the disclosure provides a sequence comprising a portion of an SV40 poly A sequence or an SV40 poly A sequence. In some cases, the disclosure provides a poly A sequence comprising a combination of one or more poly A sequences or sequence elements. In some cases, a poly A sequence is not used. In some cases, one or more poly A sequences can be referred to as an untranslated region (UTR), a 3'UTR, or a termination sequence. Preferably, an SV40 poly A sequence is used.

[0099] The polyA sequence may comprise a length of 1-10 bp, 10-20 bp, 20-50 bp, 50-100 bp, 100-500 bp, 500 bp-1 Kb, 1 Kb-2 Kb, 2 Kb-3 Kb, 3 Kb-4 Kb, 4 Kb-5 Kb, 5 Kb-6 Kb, 6 Kb-7 Kb, 7 Kb-8 Kb, 8 Kb-9 Kb and 9 Kb-10 Kb. The polyA sequence may comprise a length of at least 1 bp, 2bp, 3bp, 4bp, 5bp, 6bp, 7bp, 8bp, 9bp, 10bp, 20bp, 30bp, 40bp, 50bp, 60bp, 70bp, 80bp, 90bp, 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1 Kb, 2 Kb, 3 Kb, 4 Kb, 5 Kb, 6 Kb, 7 Kb, 8 Kb, 9 Kb and 10 Kb. The polyA sequence may comprise a length of up to 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 Kb, 2 Kb, 3 Kb, 4 Kb, 5 Kb, 6 Kb, 7 Kb, 8 Kb, 9 Kb and 10 Kb.

[0100] In some cases, the polyA sequence can be optimized for various parameters that affect protein expression, including but not limited to the mRNA half-life of the transgene in the cell, the stability of the mRNA of the transgene, or transcriptional regulation. For example, the polyA sequence can be altered to increase the mRNA transcript of the transgene, which can result in increased protein expression. In some cases, the polyA sequence can be altered to decrease the half-life of the mRNA transcript of the transgene, which can result in decreased protein expression.

[0101] In certain aspects of the present disclosure, the use of internal ribosome entry sites (IRES) or foot-and-mouth disease virus (FMDV) elements can be used to form multi-gene or multi-cistronic messages. IRES elements can bypass the ribosome scanning mode of 5' methylated Cap-dependent translation and start translation at internal sites. IRES elements from two members of the picornavirus family (poliovirus and encephalomyocarditis) have been described, and IRES from mammalian messages have also been described. IRES elements can be connected to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, thereby forming a multi-cistronic message. With the IRES element, each open reading frame can be accessible to the ribosome for efficient translation. Multiple genes can be effectively expressed using a single promoter / enhancer to transcribe a single message. An alternative system for co-expressing two proteins in a gene therapy delivery vector is the FMDV 2A system. The FMDV 2A system uses a retroviral plasmid vector in which two genes can be connected to a nucleotide sequence encoding a 2A sequence from a picornavirus foot-and-mouth disease virus. Transcription and translation produce a bicistronic mRNA and two independent protein products.

[0102] Any heterologous open reading frame can be linked to an IRES element. This can include genes for secreted proteins, multi-subunit proteins (encoded by separate genes), intracellular or membrane-bound proteins, and selectable markers. In this way, the expression of several proteins can be engineered into the cell simultaneously using a single construct and a single selectable marker.

[0103] In some aspects, the recombinant virus and / or the plasmid used to generate the recombinant virus comprises a polynucleotide encoding the human mCoChop protein or a functional fragment thereof.

[0104] In some aspects, the recombinant virus and / or the plasmid used to generate the recombinant virus comprises a regulatory nucleic acid segment capable of directing the selective expression of the mCoChop protein in eye cells.

[0105] In some aspects, the recombinant virus and / or the plasmid for generating the recombinant virus may include one or more untranslated regions (UTRs) or sequences. Generally, any suitable UTR sequence can be used for the desired optimal expression of transgenic (i.e., mCoChop). For example, in some cases, the UTR region or sequence may include a native sequence. In some cases, the UTR sequence may be a sequence visible in the upstream (5′UTR) or downstream (3′UTR) of the human CoChop gene, such as a human genome sequence or a portion thereof. In other cases, the UTR sequence may include a non-native sequence, such as a gene visible upstream or downstream of mCoChop, or include a sequence further comprising one or more UTR sequence elements, as further described herein. In some cases, only 5′UTR sequences are used. In some cases, only 3′UTR sequences are used. In some cases, UTR sequences are not used.

[0106] The UTR sequence may comprise a length of 1-10 bp, 10-20 bp, 20-50 bp, 50-100 bp, 100-500 bp, 500 bp-1 Kb, 1 Kb-2 Kb, 2 Kb-3 Kb, 3 Kb-4 Kb, 4 Kb-5 Kb, 5 Kb-6 Kb, 6 Kb-7 Kb, 7 Kb-8 Kb, 8 Kb-9 Kb, and 9 Kb-10 Kb. The UTR sequence may comprise a length of at least 1 bp, 2bp, 3bp, 4bp, 5bp, 6bp, 7bp, 8bp, 9bp, 10bp, 20bp, 30bp, 40bp, 50bp, 60bp, 70bp, 80bp, 90bp, 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1 Kb, 2 Kb, 3 Kb, 4 Kb, 5 Kb, 6 Kb, 7 Kb, 8 Kb, 9 Kb and 10 Kb. The UTR sequence may comprise a length of at most 1 bp, 2bp, 3bp, 4bp, 5bp, 6bp, 7bp, 8bp, 9bp, 10bp, 20bp, 30bp, 40bp, 50bp, 60bp, 70bp, 80bp, 90bp, 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1 Kb, 2 Kb, 3 Kb, 4 Kb, 5 Kb, 6 Kb, 7 Kb, 8 Kb, 9 Kb and 10 Kb.

[0107] In some cases, the variants of 5'UTR and / or 3'UTR can be optimized for the desired level of protein expression. In some cases, the 3'UTR sequence can be optimized for various parameters that affect protein expression, including but not limited to the mRNA half-life of the transgenic in the cell, the stability or secondary structure of the mRNA of the transgenic, or conditional regulation (e.g., the binding of various factors that regulate translation). For example, the 3'UTR sequence can be changed to increase the half-life of the mRNA transcript of the transgenic, which can result in increased protein expression. In some cases, the 3'UTR sequence can be changed to reduce the half-life of the mRNA transcript of the transgenic, which can result in reduced protein expression.

[0108] Typically, 3'UTR sequences may comprise various sequence elements. The present disclosure provides 3'UTR sequences, which may include, but are not limited to, sequence elements such as one or more polyadenylation signals, linker sequences, spacer sequences, SECIS elements, AU-rich or ARE sequences or miRNA or RNAi binding sequences, transcription termination sequences, 3' termination sequences, or variants and / or combinations thereof.

[0109] In some cases, the 5'UTR sequence can be optimized for various parameters that affect protein expression, including but not limited to the mRNA half-life of the transgene in the cell, the stability or secondary structure of the mRNA of the transgene, or transcriptional regulation. For example, the 5'UTR sequence can be altered to increase the translation efficiency of the mRNA transcript of the transgene, which can result in increased protein expression. In some cases, the 5'UTR sequence can be altered to reduce the translation efficiency of the mRNA transcript of the transgene, which can result in decreased protein expression.

[0110] Typically, 5'UTR sequences may comprise various sequence elements. The present disclosure provides 5'UTR sequences, which may include, but are not limited to, sequence elements such as one or more ribosome binding sites (RBS), linker sequences, spacer sequences, regulatory sequences, regulatory response sequences, riboswitches, sequences that promote or inhibit translation initiation, regulatory sequences for mRNA transport, or variants and / or combinations thereof.

[0111] In some aspects, the recombinant virus and / or the plasmid for generating the recombinant virus comprises one or more joints or spacer sequences. As described herein, joint sequences or spacer sequences can be used interchangeably. Generally, joint sequences or spacer sequences can be any suitable sequence for forming a discontinuous sequence between at least two sequence elements. Generally, any suitable joint or spacer sequence can be used to form a discontinuous sequence. For example, in some cases, the joint sequence can be a randomly generated sequence. In some cases, the joint sequence can be a non-specific sequence optimized to prevent the formation of secondary structures or intramolecular interactions that can adversely affect protein expression. In some cases, the joint sequence can include any additional functional sequence elements, including but not limited to introns, regulatory sequences, enhancers or similar sequences. The functional elements in the joint sequence can be used for the desired optimal generation of viruses and / or the expression of transgenic expression. In some cases, the joint sequence is a residue or other insignificant sequence of a cloning site, an existing cloning site, and the insertion of such joints between any two sequence elements is optional.

[0112] The linker sequence may comprise a length of 1-10 bp, 10-20 bp, 20-50 bp, 50-100 bp, 100-500 bp, 500 bp-1 Kb, 1 Kb-2 Kb, 2 Kb-3 Kb, 3 Kb-4 Kb, 4 Kb-5 Kb, 5 Kb-6 Kb, 6 Kb-7 Kb, 7 Kb-8 Kb, 8 Kb-9 Kb, and 9 Kb-10 Kb. The linker sequence may comprise a length of at least 1 bp, 2bp, 3bp, 4bp, 5bp, 6bp, 7bp, 8bp, 9bp, 10bp, 20bp, 30bp, 40bp, 50bp, 60bp, 70bp, 80bp, 90bp, 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1 Kb, 2 Kb, 3 Kb, 4 Kb, 5 Kb, 6 Kb, 7 Kb, 8 Kb, 9 Kb and 10 Kb. The linker sequence may comprise a length of at most 1 bp, 2bp, 3bp, 4bp, 5bp, 6bp, 7bp, 8bp, 9bp, 10bp, 20bp, 30bp, 40bp, 50bp, 60bp, 70bp, 80bp, 90bp, 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1 Kb, 2Kb, 3Kb, 4Kb, 5Kb, 6Kb, 7Kb, 8Kb, 9Kb and 10Kb.

[0113] In some aspects, the recombinant virus comprises an inverted terminal repeat (ITR) sequence for packaging the recombinant gene expression cassette into the virion of the viral vector. In some cases, the ITR is from an adeno-associated virus (AAV). In some cases, the ITR is from AAV serotype 2.

[0114] In some aspects, the recombinant virus and / or the plasmid for generating the recombinant virus comprises nucleic acid elements in the following order: a) first ITR sequence; b) enhancer sequence; c) promoter sequence; d) first exon sequence; e) intron sequence; f) second exon sequence; g) sequence encoding mCoChop; h) poly A sequence; and i) second ITR sequence. In some aspects of the recombinant virus and / or the plasmid for generating the recombinant virus, the promoter sequence comprises a promoter / enhancer sequence. In some aspects, the sequence encoding mCoChop comprises a sequence encoding a human mCoChop protein or a functional fragment thereof. In other aspects, the plasmid for generating the recombinant virus further comprises a replication origin sequence. In some aspects, the plasmid further comprises a sequence of an antibiotic resistance gene.

[0115] Pharmaceutical composition

[0116] A pharmaceutical composition is a preparation containing one or more active ingredients and one or more excipients, carriers, stabilizers or fillers, which is suitable for use in human patients to achieve desired diagnostic results or therapeutic or preventive effects. For storage stability and ease of handling, the pharmaceutical composition can be formulated as a freeze-dried (i.e., lyophilized) or vacuum-dried powder, which can be reconstituted with saline or water before the patient is administered. Alternatively, the pharmaceutical composition can be formulated as an aqueous solution. The pharmaceutical composition can contain a protein active ingredient. Various excipients such as albumin and gel have been used with varying degrees of success to dry and stably present in the protein active ingredient in the pharmaceutical composition. In addition, cryoprotectants such as alcohol have been used to reduce protein denaturation under lyophilized freezing conditions.

[0117] Pharmaceutical compositions suitable for internal use include sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be a fluid of the degree of easy injection. It must be stable under manufacturing and storage conditions, and must prevent the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing the following substances: for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and mixtures suitable therefor. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants such as polysorbate (Tween.TM.), sodium lauryl sulfate (sodium lauryl sulfate), lauryl dimethamine oxide, cetyl trimethyl ammonium bromide (CTAB), polyethoxylated alcohols, polyoxyethylene sorbitan, octoxynol (Triton X100.TM.), N,N-dimethyldodecylamine-N-oxide, cetyl trimethyl ammonium bromide (HTAB), polyethylene glycol 10 lauryl ether, Brij 721.TM., bile salts (sodium deoxycholate, sodium cholate), pluronic acid (F-68, F-127), polyoxyethylene castor oil (Cremophor.TM.), ethoxylated nonylphenol (Tergitol.TM.), cyclodextrin and ethylphenethyl ammonium chloride (Hyamine.TM.) can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.) to prevent the action of microorganisms. In many cases, it will be preferred to include isotonic agents in the composition, such as sugars, polyols (such as mannitol, sorbitol), sodium chloride. The absorption extension of the internal composition can be achieved by including agents (e.g., aluminum monostearate and gelatin) that delay absorption in the composition.

[0118] Sterile solutions can be prepared by the following: the active compound is incorporated into an appropriate solvent in the desired amount, as required, with one or more of the listed ingredients or a combination of these ingredients, and then filtered sterilized. In general, dispersions are prepared by incorporating the active compound into a sterile carrier, which contains a basic dispersion medium and other ingredients required from the above listing. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and freeze drying, which produce powders of the active ingredient and any additional required ingredients from its previously sterile filtered solution.

[0119] On the one hand, active compounds are prepared using carriers that will prevent the rapid elimination of compounds from the body, such as controlled release formulations, including implants and microencapsulation delivery systems. Biodegradable biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. The method for preparing such preparations will be clear to those skilled in the art. These materials can also be commercially available. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent number 4,522,811, which is incorporated herein by reference.

[0120] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0121] The pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids.

[0122] Certain compositions of the present disclosure also incorporate a carrier compound in the formulation. As used herein, a "carrier compound" or "carrier" may refer to a nucleic acid or an analog thereof that is inert (i.e., not biologically active per se), but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of biologically active nucleic acids by, for example, degrading biologically active nucleic acids or promoting their removal from circulation. Co-administration of nucleic acids and carrier compounds (usually an excess of the latter) may result in a significant reduction in the amount of nucleic acid recovered in the liver, kidneys, or other additional circulation reservoirs, assuming that this is due to competition between carrier compounds and nucleic acids for common receptors. For example, when a partial phosphorothioate oligonucleotide is co-administered with polycreatine, dextran sulfate, polycytidylic acid or 4-acetamido-4'isothiocyanate-diphenylethylene-2,2'disulfonic acid, the recovery of the partial phosphorothioate oligonucleotide in liver tissue may be reduced (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0123] The vector or recombinant virus (virion) can be incorporated into a pharmaceutical composition for administration to a mammalian patient, particularly a human. The vector or virosome can be formulated in a non-toxic, toxic, pharmaceutically acceptable aqueous carrier, preferably with a pH range of from 3 to 8, more preferably ranging from 6 to 8, and most preferably ranging from 6.8 to 7.2. Such a sterile composition will contain a vector or virosome containing a nucleic acid encoding a therapeutic molecule, such a composition being dissolved in an aqueous buffer having an acceptable pH upon reconstitution.

[0124] In some aspects, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of a carrier or virosome and a mixture of a pharmaceutically acceptable carrier and / or excipient such as saline, phosphate buffered saline, phosphate and amino acids, polymers, polyols, sugars, buffers, preservatives and other proteins. Exemplary amino acids, polymers and sugars, etc. are octylphenoxy polyethoxyethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, Ringer's and Hank's solutions, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinyl pyrrolidone, polyethylene and ethylene glycol. Preferably, this formulation is stable at -60°C for at least 14 months.

[0125] In some aspects, the pharmaceutical compositions provided herein comprise a buffer, such as phosphate buffered saline (PBS) or sodium phosphate / sodium sulfate, tris buffer, glycine buffer, sterile water, and other buffers known to those of ordinary skill in the art, such as those described in Good et al. (1966) Biochemistry 5: 467. Preferred pharmaceutical compositions contain sodium phosphate, sodium chloride, and sorbitol. The most preferred pharmaceutical compositions contain 10 mM sodium phosphate, 350 mM sodium chloride, and 5% (v / v) sorbitol. The pH of the buffer in which the pharmaceutical composition comprises mCoChop contained in the adenoviral vector delivery system can be in the range of 6.5 to 7.75, 6.5 to 7.5, 6.8 to 7.4, or 6.8 to 7.2.

[0126] In some aspects, the pharmaceutical compositions provided herein include substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran, in an amount of about 1-10%, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% (v / v). Preferably, sorbitol is about 3-6% (v / v), and most preferably sorbitol is about 5%. (v / v).

[0127] Prior to administration, the pharmaceutical composition is free of components used during production, such as culture components, host cell proteins, host cell DNA, plasmid DNA and is substantially free of mycoplasma, endotoxin and microbial contamination. Preferably, the pharmaceutical composition has less than 10, 5, 3, 2 or 1 CFU / swab. Most preferably, the composition has 0 CFU / swab. The endotoxin level in the pharmaceutical composition is less than 20 EU / mL, less than 10 EU / mL or less than 5 EU / mL.

[0128] The pharmaceutical composition must have a completely filled shell prior to administration. The pharmaceutical composition has a shell that is at least 50%, at least 60%, at least 70%, at least 80% or more full.

[0129] Reagent test kit

[0130] Compositions and reagents suitable for the present disclosure can be packaged in a kit to facilitate the application of the present disclosure. In some aspects, the present invention method provides a kit comprising a recombinant nucleic acid of the present disclosure. In some aspects, the present invention method provides a kit comprising a recombinant virus of the present disclosure. The instructions can be in any desired form, including but not limited to instructions printed on a kit insert, printed on one or more containers, and provided on an electronic storage medium such as a computer-readable storage medium. A software package is also optionally included on a computer-readable storage medium that allows the user to integrate information and calculate a control dose.

[0131] In another aspect, the present disclosure provides a kit comprising a pharmaceutical composition provided herein. In yet another aspect, the present disclosure provides a kit for treating a disease.

[0132] In one aspect, the kit comprises: (a) a recombinant virus as provided herein and (b) instructions for administering a therapeutically effective amount of the recombinant virus to a cell or individual. In some aspects, the kit may comprise a pharmaceutically acceptable salt or solution for administering the recombinant virus. Optionally, the kit may also comprise instructions for suitable operating parameters in the form of a label or separate insert. For example, the kit may have standard instructions for informing a doctor or laboratory technician to prepare a dose of the recombinant virus.

[0133] Optionally, the kit may further comprise standard or control information, so that the patient sample can be compared with the control information standard to determine whether the test amount of the recombinant virus is a therapeutic amount. Optionally, the kit may further comprise a device for administration, such as a syringe, a filter needle, an extension tube, a cannula, and a subretinal syringe.

[0134] Recombinant viruses can be generated by any suitable means. The methods and compositions of the present disclosure provide for generating recombinant viruses by various means, including the use of transgenic cells, which may include mammalian cells, insect cells, animal cells, or fungal cells.

[0135] For example, in some aspects, recombinant viruses can be generated by transfecting insect cells via recombinant baculovirus. In some cases, recombinant baculovirus can be generated as an intermediate, so baculovirus can contain sequences required for generating other viruses such as AAV or rAAV2 viruses. In some cases, one or more baculoviruses can be used to generate recombinant viruses for the therapeutic composition and methods of the present disclosure. In some cases, insect cells, such as Sf9, High-Five or Sf21 cell lines can be used. In some cases, cell lines can be generated using transient methods (i.e., infected with unstable integrated transgenes). In other cases, cell lines can be generated by generating stable cell lines (i.e., infected with transgenes stably integrated into the host cell genome). In other aspects, the pharmaceutical composition provided herein is manufactured using adhesive human embryonic kidney 293 (HEK293) cells. In an alternative aspect, the pharmaceutical composition provided herein is manufactured using HEK293 cells adapted to suspension. In another aspect, the pharmaceutical composition provided herein is manufactured using the baculovirus expression system (BYES) in insect cells. In some aspects, vectors are produced using herpes helper viruses. In some aspects, the vector is produced using a production cloning method. In some aspects, the vector is produced using Ad-AAV.

[0136] Generally, any suitable method can be used to biochemically purify the recombinant virus for use in a pharmaceutical composition as described herein. The recombinant virus can be harvested directly from cells or harvested from the culture medium around the host cell. The virus can be purified using various biochemical methods such as gel filtration, filtration, chromatography, affinity purification, gradient ultracentrifugation or size exclusion methods. The recombinant virus can be tested for content (i.e., consistency), purity or efficacy (i.e., activity) using any suitable method before being formulated into a pharmaceutical composition. The method can include but is not limited to immunoassay, ELISA, SDS-PAGE, Western blotting, Northern blotting, Southern blotting or PCR, HUVEC assays, etc.

[0137] Treatment

[0138] The ocular conditions that the mCoChop proteins and nucleic acids of the present invention and the resulting ChR proteins are expected to and can be used to improve one or more vision parameters include, but are not limited to, developmental abnormalities affecting the anterior and posterior segments of the eye. Anterior segment conditions include glaucoma, cataracts, corneal dystrophy, keratoconus. Posterior segment conditions include blindness conditions caused by photoreceptor failure and / or death caused by retinal dystrophy and degeneration. Retinal conditions include congenital stationary night blindness, macular degeneration such as age-related macular degeneration, congenital cone dystrophy, and a large group of retinitis pigmentosa (RP) related conditions. These conditions include the genetically predetermined death of photoreceptor cells, rods, and cones in the retina, occurring at all ages. These are severe retinal diseases that develop with age and cause blindness in childhood and early adulthood, such as subtypes of RP itself, and RP-related diseases that frequently cause vision loss during childhood, as early as one year old, such as genetic subtypes of LCA. The latter class of conditions is generally characterized by a severe reduction in photoreceptors, rods, and cones, and is often completely lost. (Trabulsi, EI ed., Genetic Diseases of the Eye, Oxford University Press, NY, 1998).

[0139] Specifically, the mCoChop and ChR proteins of the present invention are suitable for treating and / or restoring at least partial vision of a subject with vision loss due to an ocular condition such as RPE-associated retinopathy, which is characterized by long-term retention of ocular tissue structures despite loss of function, and by the correlation between loss of function and the absence or absence of normal genes in the subject's ocular cells. Various such ocular conditions are known, such as childhood onset blindness, retinitis pigmentosa, macular degeneration, diabetic retinopathy, and ocular blindness known in the art. It is expected that these other conditions and blindness conditions currently unknown and subsequently characterized by the same description as above can also be successfully treated by the CoChop and ChR proteins of the present invention. Therefore, the specific ocular conditions treated by the present invention can include the conditions described above and many diseases that have not yet been characterized.

[0140] In certain embodiments, the disclosure provides a method for treating a retinal degenerative disease, the method comprising administering a pharmaceutical composition provided herein of a pharmaceutically effective amount to a subject in need of the treatment. Preferably, the retinal degenerative disease is retinitis pigmentosa or age-related macular degeneration (AMD), wet AMD, dry AMD. In addition, other diseases and conditions that are the direct result of retinal degenerative diseases are also treated by the method of the present invention.

[0141] In some embodiments, dry AMD can be treated. In some cases, dry AMD can be referred to as central geographic atrophy, which is characterized in that the atrophy of the retinal pigment epithelium below the retina and the photoreceptor loss in the central part of the eye subsequently. Compositions and methods disclosed herein provide the treatment of any and all forms of AMD.

[0142] In a specific embodiment, the disclosure provides a method for treating AMD or retinitis pigmentosa as described herein, the method comprising administering a pharmaceutical composition provided herein to a human subject of the need for this treatment in an effective amount of medicine. The disclosure may be used to treat a patient who is at risk of developing AMD or presents the early symptoms of the disease. The disclosure may be used to treat a patient who is at risk of developing MD or presents the early symptoms of the disease, such as those individuals suffering from retinal degenerative diseases. This may include treating eyes simultaneously or in sequence. Simultaneous treatment may mean administering treatment to each eye at the same time or treating two eyes during a visit to a doctor or other medical care provider. Recorded patients have a higher risk of developing AMD in the healthy contralateral eye of the eye presenting AMD symptoms or in a patient with a genetic predisposition to develop AMD. The disclosure may be used as a preventive treatment for preventing AMD in the contralateral eye.

[0143] In some embodiments, mutant CoChop compositions disclosed herein (e.g., nucleotides, polypeptides, cells expressing the polypeptides or containing the nucleotides, pharmaceutical compositions, etc.) are administered to patients. In some embodiments, mutant CoChop compositions formed using these methods improve diseases or conditions or delay the onset of the disease or condition. In some embodiments, the disease or condition is a degenerative disease or condition. In some embodiments, the disease or condition is an eye condition. In some embodiments, the eye condition is AMD, macular degeneration, or retinitis pigmentosa. In some embodiments, the disease or condition is injury, brain injury, spinal cord injury, epilepsy, metabolic disorders, cardiac dysfunction, visual loss, blindness, deafness, hearing loss, or neurological conditions. In some embodiments, mutant CoChop compositions disclosed herein are administered to patients to restore vision loss. In some embodiments, mutant CoChop compositions disclosed herein are administered to patients to prevent, delay, or alleviate vision loss.

[0144] In some embodiments, a mutant CoChop composition disclosed herein is administered once to a patient. In some embodiments, a vector, nucleic acid, or cell disclosed herein is administered to a patient about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 20 times, about 40 times or more. A mutant CoChop composition disclosed herein is administered until the disease or condition symptoms improve.

[0145] In some embodiments, administration of a mutant CoChop composition disclosed herein improves, prevents, delays, or reduces vision loss in a treated patient compared to an untreated patient or the same patient prior to treatment. In some embodiments, administration of a mutant CoChop composition disclosed herein improves, prevents, delays, or reduces vision loss in a treated patient between day 1 and year 10. In some embodiments, administration of a mutant CoChop composition disclosed herein improves, prevents, delays, or reduces vision loss at about day 1, about day 2, about day 3, about day 4, about day 5, about day 6, about week 1, about week 2, about week 3, about week 4, about week 5, about week 6, about week 7, about week 8, about week 9, about week 10, about week 20, about week 30, about week 40, about week 50, about week 60, about week 70, about week 80, about week 90, about week 100, about year 1, about year 2, or about year 3 compared to vision loss in an untreated patient or the same patient prior to treatment. In some embodiments, administration of a mutant CoChop composition disclosed herein improves, prevents, delays, or reduces vision loss by about 1 day, about 1 week, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years or more compared to vision loss in an untreated patient or in the same patient prior to treatment.

[0146] In some embodiments, vision loss is reduced by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to a control or a patient treated with other compositions. In some embodiments, administration of a mutant CoChop composition improves, prevents, delays, or reduces vision loss by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 1 year, about 2 years, or about 3 years compared to a control or a patient treated with other compositions. In some embodiments, administration of a mutant CoChop composition disclosed herein improves, prevents, delays, or reduces vision loss by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to a control or otherwise treated patient for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years or longer.

[0147] In some embodiments, administration of a mutant CoChop composition disclosed herein increases light sensitivity in a treated patient compared to an untreated patient or the same patient prior to treatment. In some embodiments, administration of a mutant CoChop composition disclosed herein increases light sensitivity between day 1 and year 10 in a treated patient. In some embodiments, administration of a mutant CoChop composition disclosed herein increases photosensitivity at about day 1, about day 2, about day 3, about day 4, about day 5, about day 6, about week 1, about week 2, about week 3, about week 4, about week 5, about week 6, about week 7, about week 8, about week 9, about week 10, about week 20, about week 30, about week 40, about week 50, about week 60, about week 70, about week 80, about week 90, about week 100, about year 1, about year 2, or about year 3 compared to photosensitivity in an untreated patient or the same patient prior to treatment. In some embodiments, administration of a mutant CoChop composition disclosed herein increases photosensitivity by about 1 day, about 1 week, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years or more compared to photosensitivity in an untreated patient or in the same patient prior to treatment.

[0148] In some embodiments, photosensitivity is increased by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to a control or a patient treated with other compositions. In some embodiments, administration of a mutant CoChop composition increases photosensitivity by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 1 year, about 2 years, or about 3 years compared to a control or a patient treated with other compositions. In some embodiments, administration of a mutant CoChop composition disclosed herein increases photosensitivity by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years or longer compared to a control or otherwise treated patient.

[0149] In some embodiments, administration of a mutant CoChop composition disclosed herein reduces the light intensity required to induce a photocurrent in a treated patient compared to an untreated patient or the same patient prior to treatment. In some embodiments, administration of a mutant CoChop composition disclosed herein reduces the light intensity required to induce a photocurrent between day 1 and year 10 in a treated patient. In some embodiments, administration of a mutant CoChop composition disclosed herein reduces the light intensity required to elicit a photocurrent at about day 1, about day 2, about day 3, about day 4, about day 5, about day 6, about week 1, about week 2, about week 3, about week 4, about week 5, about week 6, about week 7, about week 8, about week 9, about week 10, about week 20, about week 30, about week 40, about week 50, about week 60, about week 70, about week 80, about week 90, about week 100, about year 1, about year 2, or about year 3 compared to the light intensity in an untreated patient or the same patient prior to treatment. In some embodiments, administration of a mutant CoChop composition disclosed herein reduces the light intensity required to elicit a photocurrent by about 1 day, about 1 week, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years or more compared to the light intensity required to elicit a photocurrent in an untreated patient or in the same patient prior to treatment.

[0150] In some embodiments, the light intensity required to induce a photocurrent is reduced by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to a control or a patient treated with other compositions. In some embodiments, administration of the mutant CoChop composition reduces the light intensity required to elicit a photocurrent by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 1 year, about 2 years, or about 3 years compared to a control or a patient treated with other compositions. In some embodiments, administration of a mutant CoChop composition disclosed herein reduces the light intensity required to elicit a photocurrent by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to a control or otherwise treated patient for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years or longer.

[0151] In some embodiments, administration of a mutant CoChop composition disclosed herein increases ion flux and / or proton flux in a treated patient compared to an untreated patient or the same patient prior to treatment. In some embodiments, administration of a mutant CoChop composition disclosed herein increases ion flux and / or proton flux between day 1 and year 10 in a treated patient. In some embodiments, administration of a mutant CoChop composition disclosed herein increases the ion flux and / or proton flux at about day 1, about day 2, about day 3, about day 4, about day 5, about day 6, about week 1, about week 2, about week 3, about week 4, about week 5, about week 6, about week 7, about week 8, about week 9, about week 10, about week 20, about week 30, about week 40, about week 50, about week 60, about week 70, about week 80, about week 90, about week 100, about year 1, about year 2, or about year 3 compared to the ion flux and / or proton flux in an untreated patient or the same patient prior to treatment. In some embodiments, administration of a mutant CoChop composition disclosed herein increases ion flux and / or proton flux for about 1 day, about 1 week, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years or longer compared to the ion flux and / or proton flux in an untreated patient or in the same patient prior to treatment.

[0152] In some embodiments, the ion flux and / or proton flux is increased by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to a control or a patient treated with other compositions. In some embodiments, administration of a mutant CoChop composition increases ion flux and / or proton flux by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 1 year, about 2 years, or about 3 years compared to a control or a patient treated with other compositions. In some embodiments, administration of a mutant CoChop composition disclosed herein increases ion flux and / or proton flux by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to a control or otherwise treated patient for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years or longer.

[0153] In some embodiments, administration of a mutant CoChop composition disclosed herein increases visual evoked potentials in the visual cortex in a treated patient compared to an untreated patient or the same patient prior to treatment. In some embodiments, administration of a mutant CoChop composition disclosed herein increases visual evoked potentials in the visual cortex between day 1 and year 10 in a treated patient. In some embodiments, administration of a mutant CoChop composition disclosed herein increases visual evoked potentials in the visual cortex at about day 1, about day 2, about day 3, about day 4, about day 5, about day 6, about week 1, about week 2, about week 3, about week 4, about week 5, about week 6, about week 7, about week 8, about week 9, about week 10, about week 20, about week 30, about week 40, about week 50, about week 60, about week 70, about week 80, about week 90, about week 100, about year 1, about year 2, or about year 3 compared to visual evoked potentials in the visual cortex of an untreated patient or the same patient prior to treatment. In some embodiments, administration of a mutant CoChop composition disclosed herein increases visual evoked potentials in the visual cortex for about 1 day, about 1 week, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years or longer compared to the visual evoked potentials in the visual cortex in an untreated patient or in the same patient prior to treatment.

[0154] In some embodiments, visual evoked potentials in the visual cortex are increased by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to controls or patients treated with other compositions. In some embodiments, administration of a mutant CoChop composition increases visual evoked potentials in the visual cortex by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 1 year, about 2 years, or about 3 years compared to a control or a patient treated with other compositions. In some embodiments, administration of a mutant CoChop composition disclosed herein increases visual evoked potentials in the visual cortex by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to a control or otherwise treated patient for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years or longer.

[0155] In some embodiments, administration of a mutant CoChop composition disclosed herein reduces disease or disorder symptoms in treated patients compared to untreated patients or the same patient prior to treatment. In some embodiments, these disease or disorder symptoms are measured between day 1 and year 10 in treated patients. In some embodiments, administration of a mutant CoChop composition disclosed herein reduces disease or disorder symptoms at about day 1, about day 2, about day 3, about day 4, about day 5, about day 6, about week 1, about week 2, about week 3, about week 4, about week 5, about week 6, about week 7, about week 8, about week 9, about week 10, about week 20, about week 30, about week 40, about week 50, about week 60, about week 70, about week 80, about week 90, about week 100, about year 1, about year 2, or about year 3 compared to disease or disorder symptoms in an untreated patient or the same patient prior to treatment. In some embodiments, administration of a mutant CoChop composition disclosed herein reduces disease or disorder symptoms by about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years or more compared to disease or disorder symptoms in an untreated patient or in the same patient prior to treatment.

[0156] In some embodiments, disease or condition symptoms are reduced by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to disease or condition symptoms in an untreated patient or in the same patient prior to treatment. In some embodiments, administration of a mutant CoChop composition disclosed herein reduces disease or disorder symptoms by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 1 year, about 2 years, or about 3 years compared to disease or disorder symptoms in an untreated patient or in the same patient prior to treatment. In some embodiments, administration of a mutant CoChop composition disclosed herein reduces disease or disorder symptoms by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to disease or disorder symptoms in an untreated patient or in the same patient prior to treatment for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years or longer.

[0157] In some embodiments, the administration of the mutant CoChop compositions disclosed herein reduces the symptoms of AMD in the patient treated compared to the same patient before untreated patients or treatment. In some embodiments, the symptom is blurred vision, reduced visual acuity, partial loss of vision and / or inability to see in dim light. In some embodiments, these AMD symptoms are measured between the 1st day and the 10th year in the patient treated. In some embodiments, administration of a mutant CoChop composition disclosed herein reduces AMD symptoms at about day 1, about day 2, about day 3, about day 4, about day 5, about day 6, about week 1, about week 2, about week 3, about week 4, about week 5, about week 6, about week 7, about week 8, about week 9, about week 10, about week 20, about week 30, about week 40, about week 50, about week 60, about week 70, about week 80, about week 90, about week 100, about year 1, about year 2, or about year 3 compared to AMD symptoms in an untreated patient or the same patient prior to treatment. In some embodiments, administration of a mutant CoChop composition disclosed herein reduces AMD symptoms by about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years or more compared to AMD symptoms in an untreated patient or in the same patient before treatment.

[0158] In some embodiments, AMD symptoms are reduced by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to AMD symptoms in an untreated patient or in the same patient prior to treatment. In some embodiments, administration of a mutant CoChop composition disclosed herein reduces AMD symptoms by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 1 year, about 2 years, or about 3 years compared to AMD symptoms in an untreated patient or in the same patient before treatment. In some embodiments, administration of a mutant CoChop composition disclosed herein reduces AMD symptoms by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to AMD symptoms in an untreated patient or in the same patient before treatment for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years or longer.

[0159] The terms "subject", or "individual" or "patient" as used herein refer to an individual suffering from a disease or condition or an individual suspected of suffering from a disease or condition, etc. Subject, individual or patient can be used interchangeably in the present disclosure and encompass mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the genus Mammalia: humans, non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, pigs; livestock such as rabbits, dogs and cats; laboratory animals, including rodents such as rats, mice and guinea pigs, etc. Examples of non-mammals include, but are not limited to, birds, fish, etc. In some aspects of the methods and compositions provided herein, the mammal is a human.

[0160] The efficacy of the treatment will be established, for example, by evaluating the best corrected visual acuity of each eye.Visual acuity testing is performed using the Electronic Visual Acuity (EVA) ETDRS (Early Treatment Diabetic Retinopathy Study) method or low vision assessment of hand movement and light perception.

[0161] The term "vision" as used herein is defined as the ability of an organism to usefully detect light as a stimulus for distinction or motion. Vision is intended to encompass the following:

[0162] 1. Light detection or perception - the ability to discern the presence or absence of light;

[0163] 2. Light projection - the ability to discern the direction from which light stimuli enter;

[0164] 3. Resolution - the ability to detect different brightness levels (i.e., contrast) in a grating or letter target; and

[0165] 4. Recognition - the ability to identify the shape of a visual target by referring to different levels of contrast within the target.

[0166] Therefore, "vision" includes the ability of simply detecting the presence of light. Polypeptide disclosed herein and polynucleotides encoding mutant CoChop can be used to improve or restore vision, wherein the improvement or recovery of vision includes, for example, an increase in light detection or perception, an increase in the photosensitivity or photosensitivity in response to light stimulation, an increase in the ability to distinguish the direction entered by light stimulation, an increase in the ability to detect different brightness levels, an increase in the ability to identify the shape of a visual target, and an increase in visual evoked potential or transmission from the retina to the cortex. In this way, the improvement or recovery of vision may or may not include the complete recovery of vision, i.e., wherein the vision of the patient treated with the present invention is restored to the degree of vision of the uninvolved individual. The vision recovery described in the animal study described below can be made to be at the low end of visual function by increasing an aspect of vision (i.e., photosensitivity or visual evoked potential) without restoring full vision in terms of human aspects. However, being at this level will be significantly advantageous, because these individuals can be trained in motion and potentially low-position resolution tasks, which provide them with a greatly improved level of visual independence compared to overall blindness. Even visually impaired individuals whose vision is improved using the compositions and methods of the present invention can use basic light perception to accomplish certain daily tasks and improve general movement, ability, and quality of life.

[0167] The degree of visual restoration can be determined by measuring visual acuity before and preferably after administration of a vector comprising, for example, a DNA encoding CoChop. Visual acuity can be measured using any of a number of methods well known in the art or methods yet to be determined. Visual acuity such as improved or restored by the present invention can be measured by any of the following visual responses:

[0168] 1. Light detection responses by subjects following exposure to light stimulation - where evidence is sought of a reliable response by the individual subject pointing or moving in the general direction of the light when the light is turned on;

[0169] 2. Photoprojection responses by subjects following exposure to light stimulation - where evidence of a reliable response by the individual pointing or moving in a particular light direction when the light is turned on is sought;

[0170] 3. Light discrimination of light versus dark pattern visual stimuli by the subject, which measures the subject's ability to discriminate light versus dark pattern visual stimuli, as demonstrated by:

[0171] a. Demonstrate the presence of reliable optokinetically generated nystagmus-like eye movements and / or associated head or body movements, which demonstrates tracking of the target (see above), and / or

[0172] b. the presence of a reliable ability to discriminate patterned visual stimuli and to indicate such discrimination by verbal or non-verbal means, such as pointing or pressing a lever or button; or

[0173] 4. Electrical recording of the visual cortical response to light flicker stimulation or pattern visual stimulation, which is the endpoint of electrical transmission from the recovered retina to the visual cortex, also known as visual evoked potential (VEP). Measurements can be made by electrical recordings on the scalp surface at the area of ​​the visual cortex, on the cortical surface, and / or recordings within cells of the visual cortex.

[0174] Therefore, the improvement or restoration of vision according to the present invention may include, but is not limited to: an increase in the amplitude or kinetics of the photocurrent or electrical response in response to light stimulation in retinal cells, an increase in the photosensitivity of retinal cells (i.e., a reduction in the threshold light intensity required to initiate a photocurrent or electrical response in response to light stimulation, thereby requiring less or lower light to induce a photocurrent), an increase in the number or amplitude of light-induced surges or acute excitations, an increase in the response of the retinal cortex to light, which may include an increase in visual evoked potentials transmitted from the retina or retinal cells to the visual cortex or brain.

[0175] In vitro and in vivo studies evaluating various parameters of the present invention can be used, including recognized animal models of blind human eye disorders. Large animal models of human retinopathy (e.g., childhood blindness) are useful. The examples provided herein allow those skilled in the art to easily anticipate that this method can be similarly used to treat a range of retinal diseases.

[0176] While earlier studies by others have demonstrated that retinal disease can be delayed by gene therapy techniques, the present invention demonstrates a definitive physiological restoration of function, which is expected to generate or improve various parameters of vision, including behavioral parameters.

[0177] Known animal models and tests can be used to obtain behavioral measures, such as performance in a water maze, in which subjects whose vision has been preserved or restored to varying degrees swim toward a light (Hayes, JM et al., 1993, Behav Genet 23:395-403).

[0178] In models where blindness is induced during adulthood or where congenital blindness develops slowly enough that individuals have vision prior to vision loss, subjects can be trained in various tests. In this way, when these tests are re-administered after vision loss to test the efficacy of the compositions and methods of the present invention for their vision restoration effects, the animals do not have to relearn the tasks while they were blind. Other behavioral tests do not require learning and rely on the instinctive nature of certain behaviors. An example is the optokinetic nystagmus test (Balkema GW et al., 1984, Invest Ophthalmol Vis Sci. 25:795-800; Mitchiner JC et al., 1976, Vision Res. 16:1169-71).

[0179] The present invention may also be used in combination with other forms of vision therapy known in the art to improve or restore vision. For example, a visual prosthesis is used, including a retinal implant, a cortical implant, a lateral geniculate nucleus implant, or an optic nerve implant. Therefore, in addition to genetically modifying surviving retinal neurons using the methods of the present invention, a visual prosthesis may be provided to the subject being treated before, simultaneously with, or after the molecular method is employed. The effectiveness of the visual prosthesis may be improved by training the individual, thereby enhancing the potential impact of CoChop transformation of patient cells as covered herein. Training methods, such as habituation cycles, are characterized in that the subject is trained to recognize (i) changing levels of light and / or pattern stimulation, and / or (ii) environmental stimulation from the same light or object, as will be understood by those skilled in the art; and orientation and motion training, characterized in that the subject is trained to detect visual local objects and move within the object more effectively than when not trained. In fact, any visual stimulation technique commonly used in the field of low vision restoration is applicable here.

[0180] In some embodiments, the use of different opsin genes with mutant CoChop proteins of the present invention and targeted gene expression can further increase light sensitivity or improve vision. Visual information is processed through the retina through two paths: the ON channel that sends information that the light is turned on and the OFF channel that sends information that the light is turned off. The presence of the ON channel and the OFF channel is important for contrast sensitivity. The visual signal in the ON channel is relayed from the ON-cone bipolar cells to the ON-ganglion cells. The ON-cone bipolar cells and the ON-ganglion cells depolarize in response to light. On the other hand, the visual signal in the OFF channel is carried from the OFF-cone bipolar cells to the OFF-ganglion cells. The OFF-cone bipolar cells and the OFF-ganglion cells are hypopolarized in response to light. Rod bipolar cells are responsible for the ability to see things in dim light (scotopic vision), which are ON bipolar cells (depolarized in response to light). Rod bipolar cells relay vision signals to OFF cone bipolar cells through AII type amacrine cells (ON type retinal cells).

[0181] Therefore, the dual rhodopsin system can be used to summarize the ON and OFF pathways integrated into visual processing and visual acuity. In short, the CoChop protein of the present invention can specifically target ON-type retinal neurons (i.e., ON-type ganglion cells and / or ON-type bipolar cells), while the low-polarization light sensor (i.e., halorhodopsin or other chloride pumps known in the art) can target OFF-type retinal neurons (i.e., OFF-type ganglion cells and / or OFF-type bipolar cells) to produce ON and OFF pathways. Specific targeting of preferred cell subpopulations can be achieved by using different cell type-specific promoters. For example, CoGhop expression can be driven by the mGluR6 promoter for targeted expression in ON-type retinal neurons (i.e., ON-type ganglion cells and / or ON-type bipolar cells), while low-polarization channel (such as halorhodopsin) expression is driven by the NK-3 promoter for targeted expression in OFF-type retinal neurons (i.e., OFF-type ganglion cells and / or OFF-type bipolar cells).

[0182] An alternative approach to restore ON and OFF channels in the retina is achieved by expressing depolarizing light sensors to rod bipolar cells or AII amacrine cells. In this approach, depolarization of rod bipolar cells or AII amacrine cells can induce ON and OFF responses at the level of cone bipolar cells and downstream retinal ganglion cells. Thus, the intrinsic ON and OFF channels in the retina are maintained.

[0183] Delivery Method

[0184] In some aspects, the pharmaceutical composition is administered by any method known in the art to treat or prevent a specific disease or condition. In a preferred embodiment, when treating an ocular condition, the pharmaceutical composition is administered to a site in the vitreous using any directional method. In some cases, the delivery method may be by injection, such as those described in U.S. Patent Publication No. 2010008170, which is incorporated herein by reference in its entirety. In some cases, direct administration to the vitreous includes injecting a liquid pharmaceutical composition via a syringe. In another example, direct administration may involve injection via an intubation or other suitable instruments for delivering a vector or a recombinant virus. In other examples, direct administration may include an implant, which also includes a suitable carrier for delivering a transgenic such as mCoChop. In some cases, the implant may be directly implanted in or near the retina.

[0185] Typically, the vector can be delivered as a suspension for intraocular (intravitreal) injection. Specifically, the vector is injected transscleraly through the vitreous pars plana.

[0186] definition

[0187] Unless otherwise indicated, the compositions and methods disclosed herein as described herein can be described using conventional techniques and molecular biology (including recombinant techniques), cell biology, biochemistry, immunochemistry and ophthalmological techniques, which are all within the skill of the technician practicing this technology. Such conventional techniques include methods for observing and analyzing the retina or vision in a subject, cloning and propagation of recombinant viruses, preparation of pharmaceutical compositions, and biochemical purification and immunochemistry. The specific description of suitable technology can be carried out by reference to the examples herein. However, of course, equivalent conventional procedures can also be used.Such routine techniques and descriptions can be found in standard laboratory manuals, such as Green et al., eds., Genome Analysis: A Laboratory Manual Series (Volumes I-IV) (1999); Weiner et al., eds., Genetic Variation: A Laboratory Manual (2007); Dieffenbach, Dveksler, eds., PCR Primer: A Laboratory Manual (2003); Bowtell and Sambrook, DNA Microarrays: A Molecular Cloning Manual (2003); Mount, Bioinformatics: Sequence and Genome Analysis (2004); Sambrook and Russell, Condensed Protocols from Molecular Cloning: A Laboratory Manual (2006); and Sambrook and Russell, Molecular Cloning: A Laboratory Manual (2002) (all from Cold Spring Harbor Laboratory Press); Stryer, L., Biochemistry (4th Edition) WH Freeman, NY (1995); Gait, "Oligonucleotide Synthesis: A Practical Approach" IRL Press, London (1984); Nelson and Cox, Lehninger, Principles of Biochemistry, 3rd Edition, WH Freeman Pub., New York (2000); and Berg et al., Biochemistry, 5th Edition, WH Freeman Pub., New York (2002), all of which are incorporated herein by reference in their entirety for all purposes.

[0188] Unless the context clearly indicates otherwise, as used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well. In addition, to the extent that the terms "including", "includes", "having", "has", "with" or variations thereof are used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising".

[0189] Ranges can be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another case includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation, by using the antecedent "about", it will be understood that the particular value forms another case. It will also be understood that the endpoints of each range are valid both in relation to and independently of the other endpoint. The term "about" as used herein refers to a range of plus or minus 15% from the numerical value in the context of a particular use. For example, about 10 will include a range from 8.5 to 11.5. The term "about" also describes the typical error or imprecision of the measured value.

[0190] The term "retinal degenerative disease" encompasses all diseases associated with photoreceptor degeneration. Retinal degenerative diseases include, but are not limited to, retinitis pigmentosa, age-related macular degeneration, Bart-Biedel syndrome, Bassen-Komzweig syndrome, Best disease, choroideremia, gyral atrophy, Leber congenital amaurosis, Refsun syndrome, Stargardt disease, or Usher syndrome.

[0191] In the context of the present invention, the terms "treating" or "treatment" as used herein means reversing, alleviating, inhibiting the progression of, or preventing the disorder or condition to which such terms apply, or one or more symptoms of such disorder or condition (e.g., retinal degenerative diseases).

[0192] According to the present invention, the term "patient" or "patient in need thereof" is intended for a human or non-human mammal suffering from or at risk of suffering from a retinal degenerative disease.

[0193] As intended herein, the expression "isolated nucleic acid" refers to any type of isolated nucleic acid, which may be notably natural or synthetic single-stranded or double-stranded DNA or RNA. Specifically, when the nucleic acid is synthetic, it may contain non-natural modifications of bases or bonds, particularly for increasing resistance to nucleic acid degradation. When the nucleic acid is RNA, these modifications notably encompass capping its ends or modifying the 2' position of the ribose backbone so as to reduce the reactivity of the hydroxyl moiety, for example by suppressing the hydroxyl moiety (to produce 2'-deoxyribose or 2'-deoxyribose-2'-fluororibose) or replacing the hydroxyl moiety with an alkyl group such as a methyl group (to produce 2'-O-methyl-ribose).

[0194] The term "channel rhodopsin" refers to a subfamily of sub-retinyl proteins (rhodopsins) used as light-gated ion channels. Some channel rhodopsins act as sensory light receptors in unicellular green algae, controlling phototropism: moving in response to light. Expressed in the cells of other organisms, they enable light to control electrical excitability, intracellular acidity, calcium influx and other cellular processes. They are larger than many other channel rhodopsins, with 7 transmembrane (7TM) regions and long C-terminal extensions. In green algae, they are used as visual proteins that guide algae toward or away from light sources and find the best light conditions for photosynthetic growth. The 7TM region shows some homology with other microbial (prokaryotic) rhodopsins that act as light-driven pumps (bacteriophorbosin, archaeorhodopsin and halorhodopsin) or sensors. The term also includes polypeptides homologous to channel rhodopsins.

[0195] Two amino acid sequences or nucleic acid sequences are "substantially homologous" or "substantially similar" when greater than 80%, preferably greater than 85%, preferably greater than 90% of the amino acid or nucleic acid sequences are identical or greater than about 90%, preferably greater than 95% are similar (functionally identical). In order to determine the percent identity of two amino acid sequences or two nucleic acids, these sequences are aligned for optimal comparison purposes (for example, gaps can be introduced into the sequence of the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences varies with the number of identical positions shared by the sequences. In one embodiment, the two sequences have the same length. The percent identity between the two sequences can be accomplished using a mathematical algorithm. Preferably, similar or homologous sequences are determined by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, 7th Edition, Madison, Wis.) pileup program or any sequence comparison algorithm such as BLAST, FASTA, etc.

[0196] As used herein, the term "vector" refers to a nucleic acid molecule that can transport another nucleic acid that it is connected to. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop that can be connected to other DNA segments. Another type of vector is a viral vector, in which other DNA segments can be connected to a viral genome. Some vectors can replicate autonomously in the host cell that introduces them (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (for example, non-additional mammalian vectors) are integrated into the genome of the host cell after being introduced into the host cell, and therefore replicate together with the host genome. In addition, some vectors, expression vectors can guide the expression of the gene that they are operably connected to.

[0197] Other Implementations

[0198] While the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

[0199] The patents and technical documents mentioned herein establish the knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated herein by reference in their entirety. All published foreign patents and patent applications cited herein are incorporated herein by reference in their entirety. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference in their entirety.

[0200] While the present invention has been particularly shown and described with reference to the preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made without departing from the scope of the invention as encompassed by the following claims.

[0201] The present disclosure is further illustrated by the following non-limiting examples.

[0202] Example

[0203] Example 1 - Formation and analysis of mutant CoChop polypeptides

[0204] Channelrhodopsins (ChRs) such as ChR2 are promising optogenetic light sensors for vision restoration. The main obstacle to using ChR2 for vision restoration is its low light sensitivity. We previously made more light-sensitive ChR2s by optimizing their kinetics via site-directed mutagenesis, including the most light-sensitive ChR2 mutant, ChR2-L132C / T159S. Recently, many ChR variants were reported by re-transcriptome sequencing of algae (Klapoetke et al., 2014 Nat. Methods 11(3): 338-46). We found that a variant CoChR exhibited large photocurrents. In the present invention, we made several highly light-sensitive CoChR mutants (i.e., mutant CoChop) by optimizing their kinetics via site-directed mutagenesis. These mutants include CoChR-L112C (SEQ ID NO: 3), CoChR-T139C (SEQ ID NO: 5), C68S / V69I (SEQ ID NO: 4), C68T / V69I (SEQ ID NO: 7), CoChR-T145A / S146A (SEQ ID NO: 6), CoChR-L112C / T139C (SEQ ID NO: 8), CoChR-L112C / H94E (SEQ ID NO: 9), and CoChR-L112C / H94E / K264T (SEQ ID NO: 10). CoChR and its mutants exhibit a slightly red-shifted spectral curve compared to ChR2 with a peak spectrum at 480 nm ( Figure 1 Based on electrophysiological recordings in HEK cells ( Figure 2-5 ) and multi-electrode array recordings from retinal neurons ( Figure 6 ) and optokinetic behavioral tests from blind mice ( Figure 7 ), the light sensitivity of CoChR mutants (as shown for CoChR-L112C) is much higher than the most light-sensitive ChR2 mutant ChR2-L132C / T159S. In addition, the optokinetic response of mice expressing CoChR-L112C was observed under ambient light conditions ( Figure 8 In addition, long-term stable expression of the CoChR-L112C mutant was observed in retinal neurons ( FIG. 9 ).

Claims

1. An adeno-associated virus (AAV) or recombinant adeno-associated virus (rAAV) expression vector comprising a transgene operably linked to a promoter sequence, wherein the transgene encodes a light-activated ion channel polypeptide comprising an amino acid sequence having greater than 90% amino acid sequence identity to any of SEQ ID NOs: 8, 9 or 10, comprising a cysteine ​​at position corresponding to 112 of SEQ ID NO: 2, and a cysteine ​​at position corresponding to 139 of SEQ ID NO: 2 or a glutamate at position corresponding to 94 of SEQ ID NO: 2, wherein the light-activated ion channel polypeptide, when expressed on a cell membrane and exposed to activating light, has at least one of the following: a greater level of ion flux and a greater level of proton flux compared to the light-activated ion channel polypeptide of SEQ ID NO:

2.

2. The AAV or rAAV expression vector of claim 1, wherein the transgene further comprises a threonine at a position corresponding to position 264 of SEQ ID NO:

2.

3. The AAV or rAAV expression vector of claim 1 or 2, wherein the transgene encodes a light-activated ion channel polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 8, 9 or 10, or an amino acid sequence having greater than 95% amino acid sequence identity thereto.

4. A recombinant adeno-associated virus (rAAV) expression vector comprising a transgene operably linked to a promoter sequence, wherein the transgene encodes a light-activated ion channel polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 8, 9 or 10.

5. The AAV or rAAV expression vector of claim 3, wherein the transgene encodes a light-activated ion channel polypeptide comprising the amino acid sequence of SEQ ID NO:

10.

6. A recombinant adeno-associated virus (rAAV) comprising a transgene operably linked to a promoter sequence, wherein the transgene encodes a light-activated ion channel polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 8, 9 or 10.

7. The AAV or rAAV expression vector of any one of claims 1-5, or the rAAV of claim 6, wherein the AAV or rAAV expression vector or rAAV is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12 expression vector or virus.

8. The AAV or rAAV expression vector of any one of claims 1-5, or the rAAV of claim 6, wherein the AAV or rAAV expression vector or rAAV is an AAV2 expression vector or virus.

9. The AAV or rAAV expression vector of any one of claims 1-5, or the rAAV of any one of claims 6-8, wherein the promoter is a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter, an MMT promoter, an EF-1α promoter, an UB6 promoter, a chicken β-actin promoter, a CAG promoter, an RPE65 promoter, or an opsin promoter.

10. A pharmaceutical composition comprising the AAV or rAAV expression vector of any one of claims 1-5 and 7-9.

11. A pharmaceutical composition comprising the rAAV according to any one of claims 6-9.

12. The AAV or rAAV expression vector of any one of claims 1-5 and 7-9 or the pharmaceutical composition of claim 10 or 11 for use in the preparation of a medicament for improving or restoring vision in a subject suffering from a disease or condition selected from injury, brain injury, spinal cord injury, epilepsy, metabolic disorder, cardiac dysfunction, visual loss, blindness, deafness, hearing loss or a neurological condition.

13. Use of the AAV or rAAV expression vector of any one of claims 1-5 and 7-9 or the pharmaceutical composition of claim 10 or 11 in the preparation of a medicament for improving or restoring vision.

14. The use according to claim 12 or 13, wherein the improving or restoring vision comprises any one of: increasing light sensitivity, reducing the threshold light intensity required to induce photocurrent; and increasing visual evoked potentials in the visual cortex.

15. Use of the AAV or rAAV expression vector of any one of claims 1-5 and 7-9 or the pharmaceutical composition of claim 10 or 11 in the preparation of a medicament for treating an eye disease.

16. The use according to claim 15, wherein the eye disease is retinitis pigmentosa or age-related macular degeneration.

17. Use of the AAV or rAAV expression vector of any one of claims 1-5 and 7-9 or the pharmaceutical composition of claim 10 or 11 in the preparation of a medicament for treating retinitis pigmentosa or age-related macular degeneration.

18. The use according to any one of claims 12 to 17, wherein the AAV or rAAV expression vector or the pharmaceutical composition is suitable for administration by intravitreal or subretinal injection.

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