AU2018203034A1 - rAAV-guanylate cyclase compositions and methods for treating Leber's congenital amaurosis-1 (LCA1) - Google Patents

Abstract

H \fmt\lntrwoven\NRPortbl\DCC\FMT\l0059570_1.docx-29 04 21016 Disclosed are viral vector compositions comprising polynucleotide sequences that express one or more biologically-active mammalian guanylate cyclase proteins. Also disclosed are methods for their use in preventing, treating, and/or ameliorating at least one or more symptoms of a disease, disorder, abnormal condition, or dysfunction resulting at least in part from a guanylate cyclase deficiency in vivo. In particular embodiments, the use of recombinant adeno-associated viral (rAAV) vectors to treat or ameliorate symptoms of Leber's congenital amaurosis, as well as other conditions caused by an absence or reduction in the expression of a functional retinal-specific guanylate cyclase 1 (retGC 1).

Description

DESCRIPTION

RAAV-GUANYLATE CYCLASE COMPOSITIONS AND METHODS FOR TREATING LEBER'S CONGENITAL AMAUROSIS-1 (LCA1)

BACKGROUND OF THE INVENTION

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application claims priority to United States Provisional Patent Application No. 61/327,521, filed April 23,2010, now pending, the entire contents of which is specifically incorporated herein in its entirety by express reference thereto.

[0001A] This is a divisional of Australian Patent Application No. 2016202779, which in turn is a divisional of Australian Patent Application No. 2011242527, the entire contents of which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] The United States government has certain rights in the present invention pursuant to grant numbers EY13729, EY11123, and EY08571 from the National Institutes of Health (NIH).

NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT [0003] Not Applicable.

FIELD OF THE INVENTION [0004] The present invention relates generally to the fields of molecular biology and virology, and in particular, to methods for using recombinant adeno-associated virus (rAAV) compositions that express at least a first nucleic acid segment encoding at least a first therapeutic gene product, and particularly those products useful in the prevention, treatment, or amelioration of one or more symptoms of diseases, disorders, trauma, injury, or

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2018203034 01 May 2018 dysfunction of the mammalian eye. In particular embodiments, the invention provides compositions including rAAV vectors that express a biologically-functional guanylate cyclase peptide, polypeptide, or protein for use in one or more investigative, diagnostic and/or therapeutic regimens, including, for example, the treatment of one or more disorders or diseases of the mammalian eye, and in particular, for treating congenital retinal blindness including, retinal dystrophy such as Leber’s congenital amaurosis, type 1 (LCA1), in humans. Also provided are methods for preparing rAAV vector-based guanylate cyclase medicaments for use in viral vector-based gene therapies, including, for example rAAV-LCAl vectors for treating or ameliorating one or more symptoms of guanylate cyclase deficiency in humans.

Description of Related Art [0005] Leber's congenital amaurosis (LCA) (formerly “amaurosis congenita of Leber”), first described as a congenital type of retinitis pigmentosa (RP) by German ophthalmologist Dr. Theodor Leber in 1869, is the earliest and most severe form of inherited retinopathy, and accounts for about 6% of all inherited retinal dystrophies. LCA is a group of degenerative diseases of the retina, and is the most common cause of congenital blindness in children. This autosomal recessive condition is usually recognized at birth or during the first months of life in an infant with total blindness or greatly impaired vision, normal fundus and extinguished electroretinogram (ERG) (see e.g., Perrault et al., 1996). Despite these functional deficits, LCA1 patients retain some rod and cone photoreceptors in both their macular and peripheral retina for years. Symptoms of the disease include retinal dysfunction, wobbly eye movement (nystagmus), impaired vision, slow pupil response, and ultimately, blindness.

|0006] Through genetic analyses, mutations in guanylate cyclase-1 (Gucy2d), assigned to the

LCA1 locus, have been shown to account for 20% of all reported cases of LCA (see e.g.,

Milam et ai, 2003; Perrault et al., 1996; Pen-ault et al., 2000). The number of patients affected by LCA1 is approximately twice that of patients affected by defects in the Retinal

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2018203034 01 May 2018 pigment epithelium-specific 65-kDa protein (RPE65) version of the disease (LCA2), which has garnered much attention in the gene therapy community in recent years, [0007] It is estimated that 200,000 Americans have type 1 Leber’s, Gucy2d encodes guanylate cyclase (retGCl) which is expressed in photoreceptor outer segment membranes (see e.g„ Dizhoor et al., 1994; Liu et al., 1994), and plays a role in the recovery phase of phototransduction. Mutations which reduce or abolish activity of this enzyme are thought to create the biochemical equivalent of chronic light exposure in rod and cone photoreceptors. LCA is usually regarded as the consequence of either impaired development of photoreceptors or extremely early degeneration of cells that have developed normally. The LCA1 locus (GUCY2D) has been mapped to human chromosome 17pl3.1 (LCA1) by homozygosity mapping.

Deficiencies in the Prior art

Presently there are no effective prophylactics or therapeutics available to prevent or treat LCA1 in humans.

Summary of the Invention [0008] The present invention overcomes limitations inherent in the prior art by providing new, non-obvious, and useful rAAV-based genetic constructs that encode one or more therapeutic mammalian polypeptides, and particularly those proteins, peptides, polypeptides of the guanylate cyclase family, for the prophylaxis, treatment and/or amelioration of one or more mammalian diseases, disorders or dysfunctions, or one or more symptoms thereof, that result from, or are exacerbated by, a deficit in, or a deficiency of, biologically-active guanylate cyclase polypeptide activity. In particular, the invention provides genetic constructs encoding one or more mammalian retinal-specific guanylate cyclase (retGCl) polypeptides,

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2018203034 01 May 2018 for use in the treatment of such conditions as LCA1, and other conditions of the eye such as recessive and dominant forms of cone-rod dystrophy that manifest from a deficiency or absence of physiologically-normal levels of guanylate cyclase polypeptide.

10009) In one embodiment, the invention provides a recombinant adeno-associated viral (rAAV) vector including at least a first polynucleotide that comprises a promoter operably linked to at least a first nucleic acid segment that encodes at least a first mammalian guanylate cyclase protein, peptide, or polypeptide. Preferably, the promoter is a photoreceptor-specific promoter (such as, for example, a human rhodopsin kinase promoter), or a ubiquitous promoter (such as, for example, a truncated chimeric CMV-chicken β-actin promoter). Preferably the first nucleic acid segment encodes at least a first mammalian guanylate cyclase protein, peptide, or polypeptide that comprises, consists essentially of, or alternatively, consists of, at least a first contiguous amino acid sequence region that is at least about 80%, about 85%, or about 90% or greater in identity with at least a first sequence region of at least about 60, about 70, about 80, about 90, or about 100 or more contiguous amino acids of a sequence as set forth in any one or more of the mammalian guanylate cyclase proteins depicted in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO;7, SEQIDNO:8, SEQ ID NO:9, SEQIDNOriO, or

SEQ IDNOril.

[0010] In certain embodiments, the at least a first nucleic acid segment preferably encodes at least a first mammalian guanylate cyclase protein, peptide, or polypeptide that includes at least a first contiguous amino acid sequence region that is at least about 91%, about 92%, about 93%, about 94%, or about 95% or greater in primary amino acid sequence identity with at least a first sequence region of at least about 100, about 110, about 120, about 130, about 140, or about 150 or more contiguous amino acids of a sequence as set forth in any one or more of the mammalian guanylate cyclase protein sequences recited in SEQIDNO:!,

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SEQ ID NO:2, SEQ ID N0:3, SEQ ID N0:4_} SEQ ID N0:5, SEQ ID N0:6, SEQ ID NO:7, SEQ ID NO:S, SEQ ID N0:9, SEQ ID NO:10, and SEQ ID NO: 11.

(0011J Preferably, the at least a first nucleic acid segment will encode at least one or more mammalian guanyiate cyclase proteins, peptides, or polypeptides that each preferably include at least a first contiguous primary amino acid sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to at least a first sequence region that includes at least about 90, about 110, about 130, about 150, or about 170 or more contiguous amino acids of at least a first guanyiate cyclase protein as shown in one or more of SEQ IDNO:1, SEQIDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ1DNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO;10, and

SEQ ID NO:11.

(0012) Preferably the rAAV vectors of the present invention include at least a first nucleic acid segment encodes at least a first mammalian guanyiate cyclase protein, peptide, or polypeptide that comprises, consists essentially of, or alternatively, consists of, the amino acid sequence of any one or more of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, or a polynucleotide sequence that is complementary to, or specifically hybridizes to one or more such sequences under stringent, to bighly-stringent hybridization conditions. Preferably, the first mammalian guanyiate cyclase protein, peptide, or polypeptide will possess guanyiate cyclase activity in vitro and in vivo in transformed mammalian cells, and preferably, in transformed human host cells. In particular aspects, the guanyiate cyclase protein, peptide, or polypeptide will possess significant biologically-active guanyiate cyclase activity in vitro and in vivo in transformed mammalian cells, and preferably, in transformed human host cells when the nucleic acid segment encoding the peptide, protein, or polypeptide is operably linked to at least a first promoter capable of expressing the sequence in a mammalian, and preferably, human, host cell.

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10013] While the rAAV vectors of the present invention are not necessarily limited to a particular serotype, in certain embodiments, the inventors contemplate beneficial results can be achieved by utilizing an rAAV vector that is one or more of the following known serotypes: recombinant adeno-associated virus serotype 1 (rAAV 1), recombinant adenoassociated virus serotype 2 (rAAV2), recombinant adeno-associated virus serotype 3 (rAAV3), recombinant adeno-associated virus serotype 4 (rAAV4), recombinant adenoassociated virus serotype 5 (rAAV5), recombinant adeno-associated virus serotype 6 (rAAV6), recombinant adeno-associated virus serotype 7 (rAAV7), recombinant adenoassociated virus serotype 8 (rAAV8), or a recombinant adeno-associated virus serotype 9 (rAAV) vector. In certain applications, the rAAV vectors of the present invention may be a self-complementary rAAV (scAAV) vector.

[0014] In embodiments in which a photoreceptor-specific promoter is desired, the rAAV vectors disclosed herein may include at least a first photoreceptor-specific rhodopsin kinase promoter. Exemplary such promoters include the human rhodopsin kinase promoter, which is illustrated in SEQ ID NO: 12. In certain aspects of the invention, the use of a promoter sequence that includes at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, or at least about 50 or more contiguous nucleotides from SEQ ID NO:12 is particularly preferred when tissue-specific (and in particular, photoreceptorspecific) expression of the therapeutic construct is desired.

[0015] Similarly, in embodiments in which a ubiquitous promoter is desired, the rAAV vectors disclosed herein may include at least a first truncated chimeric CMV-chicken β-actin promoter. Exemplary such promoters include the truncated chimeric CMV-chicken β-actin promoter, which is illustrated in SEQ ID NO: 13. In certain aspects of the invention, the use of a promoter sequence that includes at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, or at least about 50 or more contiguous

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2018203034 01 May 2018 nucleotides from SEQ ID NO: 13 is particularly preferred when non-tissue specific expression of the therapeutic gene is desired,

J0016] In some embodiments, the promoter sequence employed in the disclosed therapeutic gene constructs may comprise, consist essentially of, or alternately, consist of, a nucleic acid sequence that includes at least 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, or about 120 or more contiguous nucleotides from the promoter sequences set forth in either SEQ ID NO: 12 or SEQ ID NO:13.

[0017] The gene therapy vectors disclosed herein may also further optionally include one or more “upstream” or “downstream” regulatory sequences, such as a first enhancer operably linked to the at least a first nucleic segment, or a transcription regulatory region such as the woodchuck hepatitis virus post-transcriptional regulatory element. The constructs of the invention may also further optionally include one or more intron sequences operably linked to the at least a first nucleic segment encoding the therapeutic agent.

[0018] The nucleic acid segments encoding the mammalian guanylate cyclase proteins, peptides, and polypeptides of the invention may be derived from natural, semi-synthetic, or fully synthetic sequences, but will preferably be of mammalian origin. Exemplary mammalian sources include, without limitation, human, non-human primates, murines, felines, canines, porcines, ovines, bovine, equines, epine, caprine, lupines, and the like.

(0019] The rAAV vectors disclosed herein may optionally be comprised within an infectious adeno-associated viral particle, a virion, or within one or more of a plurality of infectious AAV particles. As such, the invention also emcompasses virions, viral particles, as well as isolated recombinant host cells that contain one or more of the disclosed rAAV genetic constructs. Particularly preferred host cells for the practice of the invention include, without limitation, isolated mammalian host cells that include one or more of: an rAAV vector, an

AAV virion, or a plurality of infectious viral particles.

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2018203034 01 May 2018 [00201 In other aspects, the invention provides novel and useful compositions that include one or more of (a) an rAAV vector, an rAAV virion, an rAAV infectious viral particle, a plurality of such virions or infectious particles, or an isolated mammalian host cell that comprises the vector, the virion, the infectious particle, or a plurality thereof. Preferably, such compositions will further optionally include one or more pharmaceutically-acceptable buffers, carriers, vehicles, diluents, and such like, and may further optionally include one or more lipids, liposomes, lipid complexes, ethosomes, niosomes, nanoparticles, microparticles, lipospheres, nanocapsules, or any combination thereof. Preferably such compositions are preferably formulated for administration to the human eye, and may be used in therapy or prophylaxis, and in the therapy or prophylaxis of a human retinal dystrophy, disease, or disorder (such as LCA1), in particular.

[0021| As noted below, the invention also includes diagnostic, therapeutic, and prophylactic kits that include one or more of the rAAV vector constructs disclosed herein. Such kits may further optionally include one or more protocols, dosing regimens, or instructions for using the component in the diagnosis, prevention, treatment, or amelioration of one or more symptoms of a retinal dystrophy, disease, disorder, or abnormal condition in a human. In certain aspects, therapeutic kits for the treatment of human patients diagnosed with Leber congenital amaurosis-1 (LCA-1) are particularly contemplated.

[0022J The present invention also encompasses the use of one or more of the disclosed rAAV-based compositions in therapy, or in prophylaxis of mammalian diseases or disorders. Likewise, the invention include use of the disclosed compositions in the manufacture of a medicament for diagnosing, preventing, treating or ameliorating one or more symptoms of a disease, disorder, dysfunction, or abnormal condition of a mammalian eye, and in particular, for treating or ameliorating one or more symptoms of Leber congenital amaurosis-1 (LCA-1) in a human.

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2018203034 01 May 2018 [0023] The invention also provides a method for preventing, treating or ameliorating one or more symptoms of a disease, dysfunction, disorder, deficiency, or abnormal condition in a mammal, Such method generally involves administering to a mammal in need thereof, an effective amount of an rAAV composition disclosed herein for a time sufficient to prevent, treat and/or ameliorate the one or more symptoms of the disease, dysfunction, disorder, deficiency, or abnormal condition in the mammal. Such a mammal preferably has, is suspected of having, is at risk for developing, or has been diagnosed with at least a first retinal disorder, disease, or dystrophy, including, for example, Leber congenital amaurosis-1 (LCA1), or wherein the mamma! is at risk for developing, or has been diagnosed with one or more deficiencies, defects, or absence of biologically-active, functional guanylate cyclase protein, peptide, or polypeptide. The mammal may be of any age, but will more preferably be a neonate, newborn, infant, or juvenile that is at risk for developing or has been diagnosed with a congenital retinal dystrophy such as Leber congenital amaurosis-1 (LCA-1).

[0024] The invention also further includes a method for providing a mammal with a therapeutically-effective amount of a biologically-active mammalian guanylate cyclase peptide, polypeptide, or protein to a mammal in need thereof. Such a method generally involves at least the step of introducing into suitable cells of a mamma! in need thereof, an effective amount of one or more of the rAAV vectors disclosed herein, for a time sufficient to produce a biologically-active guanylate cyclase peptide, polypeptide or protein therefrom in at least a first population of cells or at least a first tissue of the mamma into which the rAAV vector has been introduced. In the practice of the method, mammal in need thereof will preferably have one or more defects, deficiencies, or a substantial or total absence of functional, biologically-active retGCl protein in one or more tissues within or about the body ofthe mammal, when compared to the level of biologically-active retGCl protein in a normal mammal. In certain applications of the method, a plurality of cells from the mammal is provided with the rAAV vector ex vivo or in vitro, with the method further including an

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2018203034 01 May 2018 additional step of subsequently introducing the plurality of provided cells into at least a first tissue site within or about the body of the mammal. For example, the plurality of obtained cells may be introduced into at least a first site within one or both eyes of the mammal, including for example, by direct injection into the retina, the sub-retinal space, or to one or more tissues surrounding the retina, or to the entire eye, or to tissues surrounding the eye. [0025] In particular aspects, the introduction of the rAAV-vectored guanylate cyclase gene construct into the cell, and its subsequent expression permits translation of functional guanylate cyclase peptide, protein, or polypeptide, and as a result, cone photoreceptors are preserved, and cone-mediated function is restored. Importantly, such method provides for a return of normal visual behavior in the eye of the mammal, and preferably, a return of vision. [0026] Administration of the rAAV vectors of the invention may be part of a one-time therapy, or may be part of an ongoing therapy regimen repeated two or more times during the lifetime of the subject being treated. In certain aspects, a single administration of the rAAV constructs produces sustained guanylate cyclase protein formation, with preservation of the cone photoreceptors, and restoration of cone-mediated function and visual behavior over a period of at least one month, at least two months, at least three months, or longer following administration. More preferably, long-term therapy or prophylaxis is achieved using one or more subsequent administrations of the therapeutic constructs to the mammalian eye for periods of several months to several years. Preferably, cone photoreceptors are preserved, and cone-mediated function and visual behavior are restored in the mammal for a period of at least four months, at least five months, at least six months, or more following administration. In certain aspects, preservation of photoreceptors, cone-mediated function, and visual behavior are restored in the mammal for a period of at least one year, at least two years, at least three years, or at least four years or longer following completion of a treatment regimen that includes the compositions disclosed herein. Tire invention further provides a method for increasing the level of biologically-active retGCl protein in one or more retinal cells of a

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2018203034 01 May 2018 mammal that has, is suspected of having, is diagnosed with, or is at risk for developing, LCA1. Such a method generally involves introducing into at least a first population of retinal cells of a mammal in need thereof, one or more of the disclosed rAAV-guanylate cyclase viral vector constructs, in an amount and for a time effective to increase the level of biologicallyactive retGCl protein in one or more retinal cells of the mammal. Such method is particularly contemplated for preventing, treating, or ameliorating one or more symptoms of retinal dystrophy in a mammal, and may preferably involve directly or indirectly administering to the retina, sub-retinal space, or the eye of the mammal one or more of the disclosed therapeutic constructs, in an amount and for a time sufficient to treat or ameliorate the one or more symptoms of retinal dystrophy in the mammal.

|0027] The invention also provides compositions and methods for preventing, treating or ameliorating the symptoms of a guanylate cyclase protein deficiency in a mammal, and particularly for treating or reducing the severity or extent of deficiency in a human manifesting one or more of the disorders linked to a deficiency of biologically-active guanylate cyclase polypeptides. In a general sense, the method involves administration of at least a first rAAV-based genetic construct that encodes one or more guanylate cyclase peptides, polypeptides, or proteins in a phaimaceutically-acceptable vehicle to the animat in an amount and for a period of time sufficient to treat or ameliorate the deficiency in the animal suspected of suffering from such a disorder, or one or more symptoms thereof. Exemplary guanylate cyclase polypeptides useful in the practice of the invention include, but are not limited to peptides, polypeptides and proteins that have guanylate cyclase activity, and that are substantially identical in primary amino acid sequence to any one of the sequences disclosed in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQIDNO:7, SEQIDNO:S, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO:11, and to biologically-functional equivalents, or derivatives thereof. Additional exemplary guanylate cyclase peptides, proteins, and polypeptides useful in the practice of the

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2018203034 01 May 2018 include, but are not limited to those the comprise, consist essentially of, or consist of, an amino acid sequence encoding a mammalian guanylate cyclase, and particularly those sequences as disclosed in SEQIDNO;!, SEQ ID NO:2, SEQIDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQIDNO:6, SEQ ID NO:7, SEQ 1DNO:8, SEQrDNO:9, SEQ ID NO: 10, or SEQ ID NO: 11, and to biologically-functional equivalents, or derivatives thereof.

rAAV-Gcanylate Cyclase Vector Compositions |0028] In a first embodiment, the invention provides an rAAV vector comprising a polypeptide that comprises at least a first nucleic acid segment that encodes a guanylate cyclase protein, peptide or polypeptide, and in particular, a mammalian guanylate cyclase protein, peptide, or polypeptide (or a biological ly-active fragment or derivative thereof), operably linked to at least a first promoter capable of expressing the nucleic acid segment in a suitable host cell transformed with such a vector. In preferred embodiments, the nucleic acid segment encodes a mammalian, and in particular, a human, guanylate cyclase peptide, polypeptide or protein, and in particular, a peptide, polypeptide, or protein that comprises at least a first contiguous amino acid sequence that is at least 90% homologous to at least a first 30 contiguous amino acid sequence from one or more of the amino acid sequences disclosed in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ1DNO:9, SEQIDNO:10, or SEQ ID NO: 11, or a biologically-active fragment or variant thereof.

[0029) Preferably, the polypeptide comprises at least a first contiguous amino acid sequence that is at least 90%, at least 95%, or at least 98% homologous to an at least 30, an at least 40, an at least 50, an at least 60, an at least 70, or an at least 80 contiguous amino acid sequence from SEQ ID NO:1, and more preferably, the polypeptide comprises at least a first contiguous amino acid sequence that is at least 99% homologous to an at least 90 contiguous amino acid sequence from SEQ ID NO:1.

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J0030J Alternatively, the therapeutic constructs of the invention may encompass nucleic acid segments that encode guanylate cyclase polypeptides of any mammalian origin, such as for example nucleic acids, peptides, and polypeptides of murine, primate, ovine, porcine, bovine, equine, epine, caprine, canine, feline, and/or lupine origin, or may encompass modified or site-specifically mutagenized nucleic acid segments that were initially obtained from one or more mammalian species, and genetically modified to be expressed in human cells such that their guanylate cyclase activity is retained.

|0031] In other preferred embodiments, the preferred nucleic acid segments for use in the practice of the present invention, encodes a mammalian, and in particular, a human guanylate cyclase polypeptide or a biologically active fragment or variant thereof, |0032] The polynucleotides comprised in the vectors and viral particles of the present invention preferably comprise at least a first constitutive or inducible promoter operably linked to a guanylate cyclase-encoding nucleic acid segment as described herein. Such promoters may be homologous or heterologous promoters, and may be operatively positioned upstream of the nucleic acid segment encoding the guanylate cyclase polypeptide, such that the expression of the guanylate cyclase-encoding segment is under the control of the promoter. The construct may comprise a single promoter, or alternatively, two or more promoters may be used to facilitate expression of the guanylate cyclase-encoding DNA sequence.

[0033] Exemplary promoters useful in the practice of the invention include, but are in no way limited to, those promoter sequences that are operable in mammalian, and in particular, human host cells, tissues, and organs, such as for example, ubiquitous promoters, such as a CMV promoter, promoter, a β-actin promoter, a hybrid CMV promoter, a hybrid CMVβ-actin promoter, a truncated CMV promoter, a truncated β-actin promoter, a truncated hybrid CMV^-actin promoter, an EF1 promoter, a Ul a promoter, or a Ulb promoter; or one or more cell- or tissue-specific promoters (including, for example, a photoreceptor-specific promoter

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2018203034 01 May 2018 such as a rhodopsin kinase promoter [hGRKl]), or an inducible promoter such as a Tetinducible promoter or a VP16-LexA promoter.

10034] In illustrative embodiments, a polynucleotide encoding a therapeutic polypeptide was placed under the control of a ubiquitous truncated hybrid chicken β-actin (CBA) promoter, or under the control of a photoreceptor cell-specific hGRKl) promoter, and used to produce therapeutically-effective levels of the encoded guanylate cyclase polypeptide when suitable host cells were transformed with the genetic construct, and the DNA encoding the guanylate cyclase polypeptide was expressed in such cells. An example of a suitable hGRKl promoter is shown in SEQ ID NO: 12, while a suitable ubiquitous promoter, such as the truncated hybrid chicken β-actin (CBA) promoter is shown in SEQ ID NO: 13, [0035] The polynucleotides comprised in the vectors and viral particles of the present invention may also further optionally comprise one or more native, synthetic, homologous, heterologous, or hybrid enhancer or 5’ regulatory elements, for example, a natural enhancer, such as the CMV enhancer, or alternatively, a synthetic enhancer, Cell- or tissue-specific enhancers, including for example, those that increase expression of operably linked gene sequences are also contemplated to be particularly useful in the practice of the invention. Such enhancers may include, but are not limited to, retinal-specific enhancers, rod-specific enhancers, cone-specific enhancers, and such like.

[00361 The polynucleotides and nucleic acid segments comprised within the vectors and viral particles of the present invention may also further optionally comprise one or more intron sequences. In such instances, the intron sequence(s) will preferably be mammalian in origin, and more preferably, human in origin.

[0037| The DNA sequences, nucleic acid segments, and polynucleotides comprised within a vector, virion, viral particle, host cell, or composition of the present invention may also further optionally comprise one or more native, synthetic, homologous, heterologous, or hybrid posttranscriptional or 3’ regulatory elements operably positioned relative to the guanylate cyclase14

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2018203034 01 May 2018 encoding nucleic acid segments disclosed herein to provide greater expression, greater stability, and/or enhanced translation of the encoded polypeptides. One such example is the woodchuck hepatitis virus post-transcriptional regulatory element (WPR.E), operably positioned downstream of the guanylate cyclase gene. Use of elements such as these in such circumstances is well-known to those of skill in the molecular biological arts.

[0038( In illustrative embodiments, the invention concerns administration of one or more biologically-active guanylate cyclase proteins, peptides, or polypeptides that comprise an at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at about least 100, or more contiguous amino acid sequence from the polypeptide and peptide sequences disclosed hereinbelow, and particularly those polypeptides as recited in any one of SEQIDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQIDNO:4, SEQ1DNO:5, SEQ ID NO:6, SEQIDNO:7, SEQIDNO:8, SEQIDNO:9, SEQ ID NO: 10, or SEQ ID NO: 11.

[0039( Likewise, in additional illustrative embodiments, the invention concerns administration of one or more biologically-active guanylate cyclase proteins, peptides or polypeptides that are encoded by a nucleic acid segment that comprises, consists essentially of, or consists of at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about

100, at least about 110, at least about 120, at least about 130, at least about 140, at least about

150, at least about 160, at least about 170, at least about ISO, at least about 190, or at least about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, or even about 800 or more contiguous nucleic acid residues from the nucleic acid segments disclosed hereinbelow, and particularly those DNA sequences that encode any one or more mammalian guanylate cyclase proteins,

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2018203034 01 May 2018 including for example, those that are recited in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQIDNO:4, SEQ ID NO:5, SEQIDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQIDNO:9, SEQ ID NO: 10, or SEQ ID NO: 11.

[0040] Exemplary adeno-associated viral vector constructs and polynucleotides of the present invention include those that comprise, consist essentially of, or consist of at least a Erst nucleic acid segment that encodes a peptide or polypeptide that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQIDNO:!, SEQ ID NO:2, SEQ ID NO:3, SEQIDNO:4, SEQ ID NO:5, SEQIDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO:11, wherein the peptide or polypeptide has guanylate cyclase activity when expressed in selected mammalian cells and/or tissues.

[00411 In certain embodiments, the viral vector constructs and polynucleotides of the present invention will preferably include those vectors and polynucleotides that comprise, consist essentially of, or consist of at least a first nucleic acid segment that encodes a peptide or polypeptide that is at least about 82%, at least about 84%, at least about 86%, at least about 88%, at least about 92%, or at least about 94% identical to one or more of the sequences disclosed in SEQIDNO:!, SEQ1DNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQIDNO:6, SEQIDNO.7, SEQIDNO:8, SEQ ID NO:9, SEQIDNO'.IO, or SEQ ID NO: 11. Such constructs will preferably encode one or more biologically-active peptides or polypeptides that have guanylate cyclase activity when expressed in selected mammalian cells and/or tissues and in human cells and/or tissues in particular.

10042] Exemplary polynucleotides of the present invention also include those sequences that comprise, consist essentially of, or consist of at least a first nucleic acid segment that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a

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2018203034 01 May 2018 nucleic acid sequence that encodes any one of SEQ ID NO;1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQIDNOriO, or SEQ1DNO:11, wherein the peptide or polypeptide encoded by the nucleic acid segment has guanylate cyclase activity when expressed in selected mammalian cells and/or tissues.

raa V Viral Particles and Virions, and Host Cells Comprising them [00431 Other aspects of the invention concern rAAV particles and virions that comprise the rAAV-guanylate cyclase vectors of the present invention, pluralities of such particles and virions, as well as pharmaceutical compositions and host cells that comprise one or more of the rAAV-guanylate cyclase vectors disclosed herein, such as for example pharmaceutical formulations of the rAAV-guanylate cyclase vectors or virions intended for administration to a mammal through suitable means, such as, by intramuscular, intravenous, or direct injection to selected cells, tissues, or organs of the mammal, for example, one or more regions of the eye of the selected mammal. Typically, such compositions will be formulated with pharmaceutically-acceptable excipients, buffers, diluents, adjuvants, or carriers, as described hereinbelow, and may further comprise one or more liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres, or nanoparticle formulations to facilitate administration to the selected organs, tissues, and cells for which therapy is desired.

{0044] Further aspects of the invention include mammalian host cells, and pluralities thereof that comprise one or more of the rAAV vectors, virions, or infectious viral particles as disclosed herein. Particularly preferred cells are human host cells, and in particular, human ocular tissues, including, for example, retinal cells.

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Therapeutic Kits and Pharmaceutical Compositions [0045] Therapeutic kits for treating or ameliorating the symptoms of a condition resulting from a guanylate cyclase deficiency in a mammal are also part of the present invention. Exemplary kits are those that preferably comprise one or more of the disclosed AAVguanylate cyclase vector constructs, virions, or pharmaceutical compositions described herein, and instructions for using the kit. The use of such kits in methods of treatment of guanylate cyclase deficiency, and in particular, retinal-specific guanylate cyclase-1, is preferable in the treatment of retGCl defect or deficiency and in the treatment of retinal dystrophies such as

LCA-1 in an affected mammal.

]0046] Another important aspect of the present invention concerns use of the disclosed vectors, virions, compositions, and host cells described herein in the preparation of medicaments for treating or ameliorating the symptoms of guanylate cyclase deficiency in a mammal, and in particular, a human. The use of such compositions in the preparation of medicaments and in methods for the treatment of neurological and/or central nervous system defects, including for example, conditions resulting from a deficiency or defect in retinal GC1, such as for example in retinal dystrophies such as LCA-1, generally involve administration to a mammal, and particularly to a human in need thereof, one or more of the disclosed viral vectors, virions, host cells, or compositions comprising one or more of them, in an amount and for a time sufficient to treat or ameliorate the symptoms of such a deficiency in the affected mammal. The methods may also encompass prophylactic treatment of animals suspected of having such conditions, or administration of such compositions to those animals at risk for developing such conditions either following diagnosis, or prior to the onset of symptoms.

[0047] Another aspect of the invention concerns compositions that comprise one or more of the disclosed adeno-associated viral vectors, virions, viral particles, and host cells as described herein. Pharmaceutical compositions comprising such are particularly contemplated to be

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2018203034 01 May 2018 useful in therapy, and particularly in the preparation of medicaments for treating affected mammals, and humans in particular.

Therapeutic Methods [0048} The invention also provides methods for delivering therapeutically-effective amounts of a guanylate cyclase polypeptide to a mammal in need thereof. Such methods generally comprise at least the step of providing or administering to such a mammal, one or more of the guanylate cyclase compositions disclosed herein. For example, the method may involve providing to such a mammal, one or more of the rAAV vectors, virions, viral particles, host cells, or pharmaceutical compositions as described herein. Preferably such providing or such administration will be in an amount and for a time effective to provide a therapeuticallyeffective amount of one or more of the guanylate cyclase polypeptides disclosed herein to selected cells, tissues, or organs of the mammal, and in particular, therapeutically-effective levels to the cells of the mammalian eye. Such methods may include systemic injection(s) of the therapeuticum, or may even involve direct or indirect administration, injection, or introduction of the therapeutic compositions to particular cells, tissues, or organs of the mammal.

|0049| For example, the therapeutic composition may be provided to mammal by direct injection to the tissues of the eye or to the retina, or to the subretinal space, or to one or more particular cell types within the mammalian eye, (0050] The invention also provides methods of treating, ameliorating the symptoms, and reducing the severity of guanylate cyclase deficiency in an animal. These methods generally involve at least the step of providing to an animal in need thereof, one or more of the rAAV guanylate cyclase vector compositions disclosed herein in an amount and for a time effective to treat retGCl polypeptide defect or deficiency, or to treat a dysfunction resulting from such accumulation, or resulting from an underexpresison or absence of sufficient biologically19

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2018203034 01 May 2018 active guanylate cyclase polypeptide in the animal, including retinal dystrophies such as LCA I and the like. As described above, such methods may involve systemic injection(s) of the therapeuticum, or may even involve direct or indirect administration, injection, or introduction of the therapeutic compositions to particular cells, tissues, or organs of the animal.

[0051] The invention further concerns the use of the adeno-associated viral vectors, virions, viral particles, host cells, and/or the pharmaceutical compositions disclosed herein in the manufacture of a medicament for treating guanylate cyclase defect or deficiency, retinal dystrophy, or LCA1 or other GCl-related ocular disease, disorder, or dysfunction in a mammal. This use may involve systemic or localized injection, infection, or administration to one or more cells, tissues, or organs of the mammal. Such use is particularly contemplated in humans that have, are suspected of having, or at risk for developing one or more retinal dystrophies such as LCA-1.

Brief Description of the Drawings [0052| The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to the following description taken in conjunction with the accompanying drawings, in which like reference numerals identify like elements, and in which:

[0053] FIG. 1 shows representative cone- (left column) and rod- (right column) mediated ERG traces from +/+ (upper waveforms), untreated GC1 KO (middle waveforms) and AAVmGCl-treated (bottom waveforms) mice. Black traces correspond to eyes injected with hGRKl-mGCl (bottom waveforms) and their un-injected contralateral eyes (middle waveforms). Red traces correspond to eyes injected with smCBA-mGCl (bottom

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2018203034 01 May 2018 waveforms) and un-injected contralateral eyes (middle waveforms). Cone responses in AAVmGC 1 treated eyes are restored to approximately 45% of normal;

[0054] FIG. 2A and FIG. 2B show average photopic b-wave maximum amplitudes in GC1KO, isogenic +/+ controls, smCBA-mGCl-treated (FIG. 2A) and hGRKl-mGCl-treated (FIG. 2B) GC1KO mice over time. Cone responses of both smCBA-mGCl and hGRKlmGCl-treated mice are approximately 45% of normal for at least 3 months post injection; [0055J FIG. 3A, FIG. 3B, and FIG. 3C illustrate by optomotor analysis that visually-elicited behavior was restored in GCI KO mice treated with either smCBA-mGCl or hGRKl-mGCl.

Ml to M9 correspond to the nine mice used for testing. Photopic acuities and contrast sensitivities of +/+ control mice (Ml, M2), naive GC1KO (M3, M4), smCBA-mGCl (M5, M6, M7) and hGRKl-mGCl-treated (M8, M9) mice reveal that treated mice behave like normal-sighted mice (FIG. 3B and FIG. 3C). Averages of all +/+ eyes (n - 4), GC1KO eyes (n ^_ 9) and AAV-mGCl -treated eyes (n = 5) are shown (FIG. 3C), Cone-mediated ERG responses from each mouse (M1-M9) are shown for electrophysiological comparison (FIG. 3A);

10056] FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, and FIG. 4F show AAVS-hGRKlmGCl drives expression of GCI in photoreceptor outer segments of GC1KO mice (FIG. 4A). No GCI expression is seen in untreated contralateral control eye (FIG. 4B). AAV5-smCBA-mGCl drives expression of GCI in photoreceptor outer segments (FIG. 4C) and occasionally in photoreceptor cell bodes (white arrows in FIG. 4F). No such GCI expression is seen in the untreated contralateral control eye (FIG. 4D). Levels of therapeutic transgene expression in AAV5-mGCl-treated eyes are similar to that seen in isogenic +/+ control eyes (FIG. 4E). AU retinas were taken from mice 3 months’ post treatment or age matched untreated controls. Scale bars in FIG. 4A = 100 pm; in FIG, 4F = 25 pm. OS= outer segments, IS= inner segments, nuclear layer;

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2018203034 01 May 2018 [00571 FIG. 5A, FIG. 5B, FIG. 5C, and FIG. 5D show cone anrestin expression in cone photoreceptors of -+/+, GCIKO, AAV5-smCBA-mGCl-treated and AAV5-hGRKl-mGCltreated mice. Untreated GCIKO retinas contain characteristic disorganized, detached cone outer segments (FIG. 5B), whereas cone outer segments were intact and cone arrestin distribution appeared normal in treated GCIKO (FIG. 5C and FIG, 5D) and +/-+ (FIG. 5A) retinal sections. All retinas were taken from mice 3 months post treatment or age matched untreated controls. Scale bars in FIG. 5D = 100 pm, OS= outer segments, IS^- inner segments, S = synaptic terminals;

(00581 FIG. 6A, FIG, 6B, FIG. 6C and FIG. 6D show that AAV-mGCl treatment results in preservation of cone photoreceptors in treated eyes for at least three months post treatment.. Representative retinal whole mounts from the hGRKl-mGCl study (FIG. 6A: “no TX” = untreated; FIG. 6C: “TX” = treated), and the smCBA-mGCl study (FIG. 6B: “no TX = untreated; FIG. 6D: “TX” = treated) and contralateral un-injected eyes stained for cone aiTestin reveal that cone photoreceptors are preserved in GCIKO mice treated with AAVmGCl for at least 3 months post treatment. Cone cell densities were counted in central and inferior retinas of treated and untreated mice. Significant differences were found in both areas following treatment with either viral vector;

[0059] FIG. 7 illustrates the vertebrate phototransduction cascade. Upon light stimulation, conformational changes in rhodopsin (R) stimulate a cascade of events including activation of transducin (T) and cGMP phosphodiesterase (PDE) eventually resulting in the hydrolysis of cGMP. This lowering of intracellular cGMP causes a closure of the cyclic nucleotide-gated channels (CNG) in photoreceptor outer segment membranes. Closure of these channels causes hyperpolarization of the cell and therefore a dramatic drop in intracellular calcium. When calcium levels fall, unbound guanylate cyclase activating protein (GCAP) is free to stimulate guanylate cyclase (GC). GC plays a role in the recovery phase of phototransduction in that its purpose is to produce cGMP. When levels of cGMP are sufficiently increased by

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GC, cGMP-gated channels re-open causing depolarization of the cell and a return to the darkadapted state;

[0060] FIG. 8 shows the predicted structure and topology of retGCl shows homology to other guanylate cyclases with a single transmembrane spanning region, an intracellular and extracellular domain. The intracellular domain is further divided in a “kinase-like” region and catalytic domain. The calcium and GCAP1-dependent regulation of retGCl is regulated through the intracellular domains (KHD). When calcium concentration in the photoreceptor cel! is high (in the dark/depolarized state), calcium-bound GCAP1 prevents activation of retGCl. Upon light stimulation, calcium levels decrease. Calcium is unbound from GCAP1, thereby allowing GCAP1 to activate retGCl. The role of retGCl is to produce cGMP;

[0061 j FIG. 9 shows cone photoreceptors in normal (WT) v.v. GC1KO mice. In WT cones, GC1 functions normally to produce cGMP which can effectively reopen CNG gated channels and return the cell to its dark-adapted/depolarized state. In cone photoreceptors of the GC1K.0 mouse, GC1 fails to produce cGMP. This failure prevents reopening of CNG-gated channels. These cells are in essence, chronically hyperpolarized (light-adapteG). They do not transduce light for vision (as evidenced by a lack of ERG) and will eventually degenerate; [0062] FIG. 10 shows an amino acid sequence alignment of the bovine GC1 (bov GC1) and mouse GC1 (mGCl) with consensus sequence included. Variable region located in the N-terminal area is highlighted by the red rectangle;

[0063] FIG. Il shows maps of the two illustrative vectors. One contains the ubiquitous promoter smCBA, while the other utilizes the photoreceptor-specific promoter, hGRKl;

[0064] FIG. 12 shows representative retinal section from a GC1KO eye injected with AAV5smCBA-mGCl stained for GC1 (red) and PNA lectin (green) reveals GC1 expression in cone outer segments (yellow overlay) as well as in rod outer segments (red alone). bGRKl-mGCl injected eyes revealed the same pattern;

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2018203034 01 May 2018 |0065] FIG. 13A, FIG. 13B, and FIG. 13C show AAV-mediated restoration of retinal function in GC1 KO mice, FIG. 13A: Representative photopic (cone-mediated) traces recorded from eyes of GC1KO mice treated at -Pl4 with AAV5-hGRKl-mGCl (red), AAV5-smCBA-mGCl (green) or AAVS(Y733F)-hGRKl-raGCl or age-matched, isogenic GCI+/+ controls. Traces were generated at 4 months (left), 7 months (middle) and 9 months (right) post-injection, FIG. 13B: Average cone b-wave amplitudes generated monthly with a 12cds/m2 stimulus in treated GC1KO mice, untreated GC1KO and age-matched isogenic GCI+/+ control mice. FIG. 13C: Scotopic (rod-mediated) responses in treated vs. untreated GC1KO mice over time. Values represent the ratio of rod b-wave amplitudes generated at 5 cds/m2 in treated vs. untreated eyes. All three vectors confer stable, long-term therapy to GCI KO mice, with AAV8(Y733F)-hGRKl-mGCl being the most efficient;

|IH)66| FIG. 14 shows GC1KO mice treated with AAV8(Y733F)-hGRKl-mGCl were sacrificed at 7 months post injection. AAV5-smCBA-mGCl and AAV5-hGRKl-mGCltreated mice were sacrificed at 9 months post-injection. These eyes as well as that of an -11 month old GCI+/+ mice were sectioned and retinas stained with antibodies raised against GC1 (green, top row) and cone arrestin (red, bottom row). All three therapeutic vectors drove GC1 expression exclusively in photoreceptors of GC1KO mice. Some retinal thinning was observed in AAV5-hGRKl-mGCI treated mice, a result likely due to the high titer of this vector. GCI expression and cone density/morphology in AAV8(Y733F)- and AAV5smCBA- treated mice resembled that seen in age-matched GCI +/+ controls. On the contrary, retinas of an age-matched GC1KO mouse revealed an absence of GCI expression and a marked reduction in cone cell density;

10067] FIG. 15 shows at 7.5 months post-injection with AAV8(Y733F)-hGRKl-mGCl, one

GCI KO mouse was sacrificed and its retinas used for western blot. Antibodies directed against GCI show that the level of AAV-mediated GCI expression in the treated GC1KO eye are similar to that seen in the age-matched, isogenic GCI+/+ control eye. Levels of guanylate

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2018203034 01 May 2018 cyclase activating protein-1 (GCAP1) expression (a biochemical partner of guanylate cyclase) was also evaluated in treated and untreated GC1KO as well as GC1+/+ control eyes. Consistent with previous reports, GCAP1 protein was downregulated in the untreated GC1KO eyes. AAV-mediated GC1 expression results in increased GCAP1 expression, similar to levels seen in the isogenic GC1+/+ control;

[0068] FIG. 16A and FIG. 16B show results at 11 months post injection with AAV5smCBA-mGCl, one GC1KO mouse was sacrificed, its retinas whole-mounted and stained with an antibody raised against cone arrestin. The immunostain revealed tliat cones are absent in the untreated GC1KO eye (FIG. 16A) except in the superior retina. AAV-mediated GC1 expression preserves cone photoreceptors throughout the retina of the treated eye (FIG. 16B) for at least 11 months (the latest time point studied);

[0069] FIG. 17 illustrates data in which GC1KO mice injected with AAV8(733)-hGRKl mGCl were sacrificed at 4 months and 7 months post injection. GC1KO mice injected with

AAV5-smCBA-mGCl and AAV5-hGRKl-mGCl were sacrificed at 7 months and 10 months post injection. Age matched, naive GC1 KO mice were used as controls. Optic nerves from treated and untreated eyes, and portions of the right and left brains containing visual pathways were isolated and used for recovery of vector genomes. Note that AAV8(Y733F)hGRKl-mGCl was injected into the LEFT eyes of GC1K0 mice whereas both AAV5 vectors were injected into RIGHT eyes of GC1KO mice. Vector genomes were recovered only from the optic nerves of treated eyes in all cases. By 10 months post-injection of AAV5 vectors, no vector genomes were recovered from brain. The highest number of vector genomes were recovered from GC1KO mice injected with the strong, fast-acting AAV8(733) vector;

[0070] FIG. I8A and FIG. 18B illustrate data in which OCT and rod/cone ERGs from a GCdko mouse TWO months post injection with AAV8(Y733F)-hGRKl -mGCl;

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2018203034 01 May 2018 [0071] FIG. 19A and FIG. 19B illustrate data in which representative rod and cone ERGs from a GCdko mouse one month post injection with AAV8(Y733F)-hGRKl-mGCl;

[0072] FIG. 20A and FIG. 20B shows real time RT-PCR standards. Fluorescence (log units in Y-axis) is plotted against cycle threshold Crvalues (X-axis). Each panel represents the standard curves (generated by a dilution series of total retina cDNA) for GC1 and Gapdh transcript using retinal cDNA from either a wild type, GC1 +/+ mouse (a) or a GC1KO mouse treated with AAV8(Y733F)-hGRKl-mGCl. The standard curves generated by GC1 and Gapdh primer sets were parallel using either template indicating similar amplification kinetics. C_t cycle value increases with decreasing amount of template;

[0073] FIG. 21A and FIG. 21B show GC1 and cone arrestin expression in retinas of treated and untreated GC1KO mice and GC1+/+ controls. FIG. 21A: Immunohistochemistry of frozen retinal cross-sections was used to localize expression of GC 1 (green, top row) and cone arrestin (red, bottom row) in GC1KO mice treated with AAV8(Y733F)-hGRKl-mGCl (7 months post-inject!on), AAVS-smCBA-mGCl (10 months post-injection) or AAV5-hGRKlmGCI (10 months post-injection) vectors as well as retinas from 8 month old untreated GC1 KO and GC1+/+ control mice. Nuclei were stained with DAP1 (blue). All sections were imaged at 20X magnification and exposed at identical settings. FIG. 2IB: Immunostaining of retinal whole mounts from one GC1 KO mouse 11 months post-treatment with AAVSsmCBA-mGCl (one eye only) with an antibody against cone arrestin revealed marked preservation of cone photoreceptors in the treated eye (bottom right) compared to the untreated contralateral control eye (bottom left). Retinal whole mounts were oriented similarly, with their temporal portions in the 12 o’clock position. Portions of whole mounts were imaged at 1 OX magnification and merged together for final presentation. OS= outer segments; nuclear layer; INL= inner nuclear layer;

]0074] FIG. 22A and FIG. 22B show cone-mediated electroretinograms (ERGs) of treated and untreated GC1KO and untreated GC1+/+ control eyes, FIG. 22A: Representative cone26

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2018203034 01 May 2018 mediated traces elicited by a 12 cds/m² light stimulus from GC1K0 eyes treated with AAV5hGRKl-mGCl (red line), AAV5-smCBA-mGCl (green line) or AAV8(Y733F)-hGRKlmGCl (black line) or untreated age-matched GC1+/+ control eyes. Representative traces generated between 4-months’ and I-years’ post-treatment are shown (top panel). Scale: yaxis = 50 pV, x-axis = 20 ms. FIG. 22B: Maximum cone b-wave amplitudes (those generated at 12 cds/m²) were calculated from each mouse and averaged monthly in each treatment group as well as age-matched, untreated GC1KO and GC 1+/+- controls. Comparisons were made between groups of animals with an n > 3. All AAV treatment groups were statistically compared for 6-months’ post-treatment. AAV5 vector treated eyes were statistically compared for 9-months’ post-treatment;

[0075] FIG. 23 A and FIG, 23B illustrate rod-mediated electroretinograms (ERGs) of treated and untreated GC1KO and GC 1+/+ control eyes. FIG.23A: Rod b-wave amplitudes (top left) and a-wave amplitudes (top right) elicited by a 1 cds/m² stimulus under scotopic conditions were determined in the treated and untreated eyes of GC1K.0 mice treated with AAV8(Y733F)-hGRKl-mGCl (black circles), AAV5-hGRKl-mGCl (red circles) or AAV5smCBA-mGCl (green triangles) vector. Intra-mouse ratios of treated and untreated eyes were generated by dividing the maximum a- or b-wave amplitude in treated eyes by the maximum amplitude in the untreated eye. These ratios were averaged monthly in all treatment groups. Comparisons were made between groups of animals with an n > 3. All AAV treatment groups were statistically compared for 6 months. AAV5 vectors were also statistically compared for 9 months. Vector-mediated improvement was defined by an average ratio >0.8. FIG. 23B: Representative rod-mediated ERG traces from one GC1KO mouse reveal that rod responses from the AAV8(Y733F)-hGRKl-mGCl-treated eye (black line) were higher than those recorded from the untreated contralateral control eye (green line). This treated rod response was restored to ~50% that of the normal GC1+/+ rod response (red line);

and

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2018203034 01 May 2018 fO076| FIG. 24A and FIG. 24B show protein and transcript levels in treated and untreated GC1KO mice and GC1+/+ controls. FIG. 24A: ImmunobJot of retinal lysates from one GC1KO mouse eye at 10 months after treatment with AAV8(Y733F)-hGRKl-mGCl and probed with anti-GCl and anti-GCAPl antibodies, Αηίί-β-actin antibody was used as an internal loading control. FIG. 24B: Semiquantitative real time RT-PCR of several transcripts (GC1, GCAP1, GNAT2, and PDE6a in one GC1KO retina treated with AAV5-smCBAmGCl, one GC1 KO retina treated with AAV8(Y733F)-hGRKl-mGCl vector, and in individual untreated GC1KO or GC1+/+ control retinas. Samples were performed in triplicate using Gapdh-specific primers as a standard. Data is presented as the fold-change in mRNA levels relative to the GC1+/+ control.

Description of Illustrative Embodiments [0077] Illustrative embodiments of the invention are described below. In the interest of clarity, not all features of an actual implementation are described in this specification. It will of course be appreciated that in the development of any such actual embodiment, numerous implementation-specific decisions must be made to achieve the developers’ specific goals, such as compliance with system-related and business-related constraints, which will vary from one implementation to another. Moreover, it will be appreciated that such a development effort might be complex and time-consuming, but would nevertheless be a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure.

Adeno-Associated Virus [0078] Adeno-associated virus-2 (AAV) is a human parvovirus that can be propagated both as a lytic virus and as a provirus (Cukor et ai, 1984; Hoggan et al., 1972). The viral genome consists of linear single-stranded DNA (Rose etal., 1969), 4679 bases long (Srivastava et al.,

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1983) , flanked by inverted terminal repeats of 145 bases (Lusby etal., 1982). For lytic growth AAV requires co-infection with a helper virus. Either adenovirus (Atchinson etal., 1965; Hoggan, 1965; Parks etal., 1967) or herpes simplex (Buller etal., 1981) can supply helper function. Without helper, there is no evidence of AAV-specific replication or gene expression (Rose and Koczot, 1972; Carter etal., 1983). When no helper is available, AAV can persist as an integrated provirus (Hoggan, 1965; Bems etal., 1975; Handa etal., 1977; Cheung et al., 1980; Bems et al., 1982),

10079J Integration apparently involves recombination between AAV termini and host sequences and most of the AAV sequences remain intact in the provirus. The ability of AAV to integrate into host DNA is apparently an inherent strategy for insuring the survival of AAV sequences in the absence of the helper virus. When cells carrying an AAV provirus are subsequently superinfected with a helper, the integrated AAV genome is rescued and a productive lytic cycle occurs (Hoggan, 1965), (0080] AAV sequences cloned into prokaryotic plasmids are infectious (Samulski etal., 1982). For example, when the wild type AAV/pBR322 plasmid, pSM620, is transfected into human cells in the presence of adenovirus, the AAV sequences are rescued from the plasmid and a normal AAV lytic cycle ensues (Samulski et al., 1982). This renders it possible to modify the AAV sequences in the recombinant plasmid and, then, to grow a viral stock of the mutant by transfecting the plasmid into human cells (Samulski et al., 1983; Hermonat et a!.,

1984) , AAV contains at least three phenotypically distinct regions (Hermonat et al., 1984), The rep region codes for one or more proteins that are required for DNA replication and for rescue from the recombinant plasmid, while the cap and lip regions appear to code for AAV capsid proteins and mutants within these regions are capable of DNA replication (Hermonat et al., 1984). It has been shown that the AAV termini are required for DNA replication (Samulski etal., 1983).

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2018203034 01 May 2018 (0O81J Laughlin et al. (1983) have described the construction of two E. coli hybrid plasmids, each of which contains the entire DNA genome of AAV, and the transfection of the recombinant DNAs into human cell lines in the presence of helper adenovirus to successfully rescue and replicate the AAV genome (See also Tratschin et ai, 1984a; 1984b).

[0082] Adeno-associated virus (AAV) is particularly attractive for gene transfer because it does not induce any pathogenic response and can integrate into the host cellular chromosome (Kotin et ai, 1990). The AAV terminal repeats (TRs) are the only essential cis-components for the chromosomal integration (Muzyczka and McLaughin, 1988). These TRs are reported to have promoter activity (Flotte etai, 1993). They may promote efficient gene transfer from the cytoplasm to the nucleus or increase the stability of plasmid DNA and enable longerlasting gene expression (Bartlett and Samulski, 1998), Studies using recombinant plasmid DNAs containing AAV TRs have attracted considerable interest. AAV-based plasmids have been shown to drive higher and longer transgene expression than the identical plasmids lacking the TRs of AAV in most cell types (Philip el al., 1994; Shafron et ai, 1998; Wang et ai, 1998).

(O083J There are several factors that prompted researchers to study the possibility of using rAAV as an expression vector. One is that the requirements for delivering a gene to integrate into the host chromosome are surprisingly few. It is necessary to have the 145-bp ITRs, which are only 6% of the AAV genome. This leaves room in the vector to assemble a 4,5-kb DNA insertion. While this carrying capacity may prevent the AAV from delivering large genes, it is amply suited for delivering the antisense constructs of the present invention.

(00841 AAV is also a good choice of delivery vehicles due to its safety. There is a relatively complicated rescue mechanism: not only wild type adenovirus but also AAV genes are required to mobilize rAAV. Likewise, AAV is not pathogenic and not associated with any disease. The removal of viral coding sequences minimizes immune reactions to viral gene expression, and therefore, rAAV does not evoke an inflammatory response. AAV therefore,

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2018203034 01 May 2018 represents an ideal candidate for delivery of the guanyiate cyclase-encoding polynucleotides of the present invention.

Production of rAAV Vectors [0085J Traditional protocols to produce rAAV vectors have generally been based on a threecomponent system. One component of this system is a proviral plasmid encoding the recombinant DNA to be packaged as rAAV. This recombinant DNA is located between 145 base pair (bp) AAV-2 inverted terminal repeats (ITRs) that are the minimal cis acting AAV-2 sequences that direct replication and packaging of the vector. A second component of the system is a plasmid encoding the AAV-2 genes, rep and cap. The AAV-2 rep gene encodes four Rep proteins (Rep 78, 68, 52 and 40) that act in trans to replicate the rAAV genome, resolve replicative intermediates, and then package single-stranded rAAV genomes. The AAV-2 cap gene encodes the three structural proteins (VP1, VP2, and VP3) that comprise the virus capsid. Because AAV-2 does not proficiently replicate on its own, the third component of a rAAV packaging system is a set of helper functions from another DNA virus. These helper functions create a cellular environment in which rAAV replication and packaging can efficiently occur. The helper functions provided by adenovirus (Ad) have almost exclusively been used to produce rAAV and are encoded by the genes El a, Elb, E2a, E4orf6, and VA RNA. While the first two components of the system are generally introduced into cells in which replication and packaging is to occur by transfection, ad helper functions are introduced by superinfection with wild type Ad virus.

[0086] The traditional rAAV production techniques are limited in their ability to produce large quantities of vector because of inherent inefficiencies in transfection. Serious difficulties are also encountered when the scale of transfection is increased. The requirement for wild type Ad may also reduce the amount of rAAV produced since Ad may compete for cellular and viral substrates that are required for viral replication but are present only in

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2018203034 01 May 2018 limiting amounts. Another problem encountered in traditional production protocols is that superinfection with Ad requires development of effective procedures for purification of Ad from the rAAV produced. While these purification processes are generally successful at eliminating Ad contamination of rAAV preparations, they also reduce rAAV titers. Stringent assays for Ad contamination of rAAV are also necessary.

[0087) To produce rAAV, a double co-transfection procedure is used to introduce a rAAV transfer vector plasmid together with pDG (Grimm et al., 1998) AAV helper plasmid carrying the AAV rep and cap genes, as well as Ad helper genes required for rAAV replication and packaging at a 1:1 molar ratio. Plasmid DNA used in the transfection is purified by a conventional alkaline lysis/CsCl gradient protocol. The transfection is carried out as follows: 293 cells are split 1:2 the day prior to the experiment, so that, when transfected, the cell confluence is about 75-80%. Ten 15-cm plates are transfected as one batch. To make CaPO4 precipitate 0.7 mg of pDG are mixed with 180 pg of rAAV transfer vector plasmid in a total volume of 12.5 mL of 0.25 M CaC12. The old media is removed from the cells and the formation of the CaPO4-precipitate is initiated by adding 12.5 ml of 2X HBS (pH 7.05) that has been pre-wanned to 37°C to the DNA-CaC12 solution. The DNA is incubated for 1 min; and transferring the mixture into 200 mL of pre-warmed DMEM-10% FBS then stops the formation of the precipitate. Twenty two mL of the medium is immediately dispensed into each plate and cells are incubated at 37°C for 48 hr. The CaPO4-precipitate is allowed to stay on the cells during the whole incubation period without compromising cell viability. Fortyeight hr post-transfection cells are harvested by centrifugation at 1,140 x g for 10 min. Cells are then lysed in 15 ml of 0.15 M MgCl, 50 mM Tris-HCl (pH 8.5) by 3 frecze/thaw cycles in dry ice-ethanol and 37°C baths. Benzonase (Nycomed Pharma A/S, pure grade) is added to the mixture (50 U/mL final concentration) and the lysate is incubated for 30 min at 37°C. The lysate is clarified by centrifugation at 3,700 xg for 20 min and the virus-containing supernatant is further purified using a discontinuous density gradient.

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2018203034 01 May 2018 [0088] The typical discontinuous step gradient is formed by underlayering and displacing the less dense cell lysate with Iodixanol, 5,5[(2-hydroxi-l-3-propanediyl)-bis(acetylamino)] bis [N,Nbi ,(2,3dihydroxypropyl-2-4,6-triiodo-l,3-enzenecarboxamide], prepared using a 60% (wt/vol.) sterile solution of OptiPrep (Nycomed). Specifically, 15 mL of the clarified lysate are transferred into Quick-Seal Ultra-Clear 25 x 89-mm centrifuge tube (Beckman) using a syringe equipped with a 1/27 ^x 89 mm spinal needle. Care is taken to avoid bubbles, which would interfere with subsequent filling and sealing of the tube. A variable speed peristaltic pump, Model EP-1 (Bio-Rad), is used to underlay in order: 9 mL of 15% iodixanol and 1 M NaCl in PBS-MK buffer containing Phenol Red (2.5 pL of a 0.5% stock solution per ml of the iodixanol solution); 5 mL of 40% iodixanol in PBS-MK buffer; and finally, 5 mL of 60% iodixanol in PBS-MK buffer containing Phenol Red (0.1 pL/L), Tubes are sealed and centrifuged in a Type 70 Ti rotor (Beckman) at 350,000 ^x g for 1 hr at 18°C, Four mL of the clear 40% step is aspirated after puncturing the tube on the side with a syringe equipped with an 18-gauge needle with the bevel uppermost. The iodixanol fraction is further purified using conventional Heparin agarose affinity chromatography.

(0089] For chromatography, typically, a pre-packed 2.5-mL Heparin agarose type I column (Sigma) is equilibrated with 20 mL of PBS-MK under gravity. The rAAV iodixanol fraction is then applied to the pre-equilibrated column, and the column is washed with 10 mL of PBSMK. rAAV is eluted with the same buffer containing 1 M NaCl. After applying the elution buffer, the first 2 ml of the eluant are discarded, and the virus is collected in the subsequent

3.5 mL of elution buffer.

[0090] Virus is then concentrated and desalted by centrifugation through the BIOMAX® 100 K filter (Millipore, Bedford, MA, USA) according to the manufacturer’s instructions. The high salt buffer is changed by repeatedly diluting the concentrated virus with Lactated Ringer’s solution, and repeating the titer both genome containing particles and infectious rAAV particles, A conventional dot-blot assay, quantitative competitive PCR (QC PCR)

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2018203034 01 May 2018 assay, or more recently quantitative real-time PCR (aRT-PCR) are used to determine physical particle titers (Zolotukhin et al., 2002; Jacobson et al., 2006) Infectious titers are determined by infectious center assay (ICA) and fluorescent cell assay (FCA), which scores for expression of GFP (Zolotukhin et al., 2002).

|00911 QC PCR method is based on competitive co-amplified of a specific target sequence with internal standard plasmid of known concentration in on reaction tube. It provides precise and fast quantitation of viral particles. The internal standard must hare primer recognition sites with the specific template. Both the specific template and the internal standard must be PCR-ampfified with the same efficiency and it must be possible to analyze the PCR-amplified products separately. The easiest way to distinguish between the template and the internal standard is to incorporate a size difference in the two products. This can be achieved, for example, by constructing standards having the same sequence as the specific target but containing a deletion. Quantitation is then performed by comparing the PCR signal of the specific template with the PCR signal obtained with known concentrations of the competitor (the internal standard). Quantitative real-time PCR (qRT-PCR) is a standard method for evaluating DNA concentration of an unknown sample by comparison of PCR product formation in real-time to a known DNA standard.

(0092] The purified viral stock is first treated with DNAsel to digest any contaminating unpackaged DNA. Ten pL of a purified virus stock is incubated with 10 U of DNAsel (Boehringer, Ingelheim am Rhein, Germany) in a 100 pL reaction mixture, containing 50 mM

Tris-HCl (pH 7.5), 10 mM MgCl₂ for 1 hr at 37°C, At the end of the reaction, 10 pL of 10X

Proteinase K buffer (10 mM Tris-HCl [pH 8.0], 10 mM EDTA, 1% SDS final concentration) was added, followed by the addition of 1 pL of Proteinase K (18.6 mg/mL, Boehringer). The mixture was incubated at 37°C for 1 hr. Viral DNA was purified by phenol/chloroform extraction (twice), followed by chloroform extraction and ethanol precipitation using 10 pg of glycogen as a carrier. The DNA pellet was dissolved in 100 pL of water. QC PCR reaction 34

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2018203034 01 May 2018 mixtures each contained 1 pL of the diluted viral DNA and two-fold serial dilutions of the internal standard plasmid DNA, such as pdl-GFP. The most reliable range of standard DNA was found to be between 1 and 100 pg. An aliquot of each reaction was then analyzed by 2% agarose gel electrophoresis, until two PCR. products were resolved. The analog image of the ethidium bromide stained gel was digitized using and hnageStore 7500 system (UVP; Upland, CA, USA). The densities of the target and competitor bands in each lane were measured using the ZERO-Dscan Image Analysis System, version 1.0 (Scanalytics, Rockville, MD, USA) and their ratios are plotted as a function of the standard DNA concentration. A ratio of 1,0, at which the number of viral DNA molecules equals the number of competitor DNA molecules was used to determine the DNA concentration of the virus stock.

)0093) A modification of the previously published protocol (McLaughlin et al., 1988) was used to measure the ability of the virus to infect C12 cells, unpackage, and replicate. Briefly, C2 cells containing integrated wtAAV rep and cap genes (Clark et al., 1995) were plated in a 96-well dish at about 75% confluence, then infected with Ad5 at a M.O.I of 20. One pL of serially diluted rAAV-sCNTF was visually scored using a fluorescence microscope. High sensitivity CHROMA filter #41012 HighQ FITC LP (Chroma Technology, Bellows Fall, VA, USA) was used to monitor the fluorescence. To calculate the titer by hybridization, cells were harvested and processed essentially as previously described (McLaughlin et al., 1988),

Pharmaceutical Compositions [0094] In certain embodiments, the present invention concerns formulation of one or more of the rAAV-guanylate cyclase compositions disclosed herein in pharmaceutically acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy.

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2018203034 01 May 2018 [0095j Π will also be understood that, if desired, the nucleic acid segment, RNA, DNA or PNA compositions that express a therapeutic gene product as disclosed herein may be administered in combination with other agents as well, such as, e.g., proteins or polypeptides or various pharmaceutically-active agents, including one or more systemic or topical administrations of guanylate cyclase polypeptides. In fact, there is virtually no limit to other components that may also be included, given that the additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues. The rAAVvectored guanylate cyclase compositions may thus be delivered along with various other agents as required in the particular instance. Such compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesized as described herein. Likewise, such compositions may further comprise substituted or derivatized RNA, DNA, or PNA compositions.

[0096J Formulation of pharmaceutically-acceptable excipients and carrier solutions is wellknown to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, intramuscular, and direct administration to one or more cells or tissue types within the animal, including for example, ocular, retinal, and sub-retinal injection or such like.

[0097] Typically, these formulations may contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 60% or 70% or more of the weight or volume of the total formulation. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one of ordinary skill in the art of

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2018203034 01 May 2018 preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0098] In certain circumstances it will be desirable to deliver the pharmaceutical compositions disclosed herein parenterally, intravenously, intramuscularly, or even intraperitoneally as described (see e.g., U. S. Patent No. 5,543,158; U. S. Patent No, 5,641,515 and U. S. Patent No. 5,399,363, each of which is specifically incorporated herein in its entirety by express reference thereto). Solutions of the active compounds as freebase or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. [0099| The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (see e.g., U. S. Patent No. 5,466,468, specifically incorporated herein in its entirety by express reference thereto). In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can he a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may 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 dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial ad antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can

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2018203034 01 May 2018 be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin, [00100] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCI solution and either added to 1000 mL of hypodermoclysis fluid or injected at the proposed site of infusion, (see, e.g., Remington’s Pharmaceutical Sciences 15th Ed., pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and the general safety and purity standards as required by the United States Food and Drug Administration’s (FDA) Office of Biologies Standards.

[00101] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients as enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

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2018203034 01 May 2018 [00102] The compositions disclosed herein may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.

100103] As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

100104] The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified.

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Sequence Comparison, Identity, and Homology

100105] For sequence comparison and homology determination, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm can then be used to calculate the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[00106] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm (see e.g., Smith and Waterman, 1981), by the homology alignment algorithm (see e.g., Needleman and Wunsch, 1970), by the search similarity comparison method (see e.g., Pearson and Lipman, 1988), by computerized implementations of algorithms such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI, USA, or by visual inspection. One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm (Altschul etal.. 1990) and BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff, 1989). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih. gov/), [00107] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g,, Karlin and Altschul, 1993). Another example of a useful sequence alignment algorithm is the PILEUP program, which creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment, PILEUP uses a simplification of the progressive

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2018203034 01 May 2018 alignment comparison method (see e.g., Feng and Doolittle, 1987), and employs a general alignment matrix similar to that described by Higgins and Sharp (1989).

Therapeutic and Diagnostic Kits [00108) The invention also encompasses one or more compositions together with one or more pharmaceutical] y-acceptable excipients, carriers, diluents, adjuvants, and/or other components, as may be employed in the formulation of particular rAAV-guanylate cyclase formulations, and in the preparation of therapeutic agents for administration to a mammal, and in particularly, to a human, for one or more of the guanyiate cyclase-deficient conditions, such as a retinal dystrophy like LCA1, as described herein. In particular, such kits may comprise one or more rAAV-vectored guanyiate cyclase composition in combination with instructions for using the viral vector in the treatment of such disorders in a mammal, and may typically further include containers prepared for convenient commercial packaging.

|00109] As such, preferred animals for administration of the pharmaceutical compositions disclosed herein include mammals, and particularly humans. Other preferred animals include non-human primates, murines, epines, bovines, ovines, equines, hircines, lupines, leporines, vulpines, porcines, canines, felines, and the like. The composition may include partially or significantly purified rAAV-guanylate cyclase compositions, either alone, or in combination with one or more additional active ingredients, which may be obtained from natural or recombinant sources, or which may be obtainable naturally or either chemically synthesized, or alternatively produced in vitro from recombinant host cells expressing DNA segments encoding such additional active ingredients.

|00110| Therapeutic kits may also be prepared that comprise at least one of the compositions disclosed herein and instructions for using the composition as a therapeutic agent. The container means for such kits may typically comprise at least one vial, test tube, flask, bottle, syringe or other container means, into which the disclosed rAAV composition(s) may be

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2018203034 01 May 2018 placed, and preferably suitably aliquotted. Where a second guanylate cyclase composition is also provided, the kit may also contain a second distinct container means into which this second composition may be placed. Alternatively, the plurality of guanylate cyclase compositions may be prepared in a single pharmaceutical composition, and may be packaged in a single container means, such as a vial, flask, syringe, bottle, or other suitable single container means. The kits of the present invention will also typically include a means for containing the vial(s) in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vial(s) are retained.

Expression in Animal Cells [00111] The inventors contemplate that a polynucleotide comprising a contiguous nucleic acid sequence that encodes a therapeutic guanylate cyclase polypeptide of the present invention may be utilized to treat one or more cellular defects in a transformed host cell. Such cells are preferably animal cells, including mammalian cells such as those obtained from a human or a non-human primate, or from one or more mammalian species including without limitation, murines, canines, bovines, equines, epines, felines, ovines, hircines, lupines, leporines, porcines, and the like. The use of such constructs for the treatment and/or amelioration of one or more symptoms of a retinal dystrophy such as LCA1, or of a related retinal or ocular disease, disorder, condition, or dysfunction in a human subject suspected of suffering from such a disorder, or at risk for developing such a condition is particularly contemplated by the present inventors, [00112] The cells may be transformed with one or more rAAV vectors comprising one or more therapeutic guanylate cyclase genes of interest, such that the genetic construct introduced into and expressed in the host cells of the animal is sufficient to alter, reduce, ameliorate or prevent the deleterious or disease condition(s) or one or more symptoms thereof, either ex vivo, in vitro, ex situ, in situ, and/or in vivo.

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Guanylate Cyclase (00113) Guanylate cyclase (GC) (EC 4.6.1.2) is a lyase that catalyzes the conversion of guanosine triphosphates (GTP) to 3', 5'-cyclic guanosine monophosphate (cGMP) and pyrophosphate. Referred to alternatively in the literature as “guanyl cyclase” or “guanylyl cyclase,” both membrane-bound (type 1) and soluble (type 2) forms of GC exist.

Leber Congenital Amaurosis [00114| Leber congenital amaurosis (LCA) is an autosomal recessive group of diseases that represent the earliest and most severe form of all inherited retinal dystrophies. The first gene implicated in the onset of this genetically and clinically heterogeneous disease, and therefore assigned to the LCA1 locus was retinal-specific Guanylate cyclase-1 (Gucy2d) (Perrault et al., 1996), Gucy2d encodes for the retinal specific protein guanylate cyclase (retGCl) which is expressed predominantly in photoreceptor outer segment membranes and plays a role in the regulation of cGMP and Ca2+ levels within these cells. Following light stimulation, levels of cGMP within photoreceptor outer segments rapidly fall due to hydrolysis by cGMP phosphodiesterase (PDE). This reduction of cGMP leads to a closure of cGMP-gated channels, reduced Ca2+ influx, and hyperpolarization of the cell. This decrease in intracellular Ca2+ stimulates recovery of light-stimulated photoreceptors to the dark state via its interaction with guanylate cyclase activating proteins (GCAPs), a family of calcium binding proteins that regulate the activity of GC. In the dark adapted photoreceptor, Ca2+-bound GCAPs inhibit the activity of GC. Upon light stimulation, however, Ca2+-free GCAPs stimulate GC activity which produces an increase in cGMP levels, a reopening of the cGMP-gated channels and a return of the cell to a depolarized state. Mutations which reduce or abolish the ability of GC to replenish intracellular cGMP and reopen cGMP-gated cation

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2018203034 01 May 2018 channels, as is the case in LCA1, are thought to create the biochemical equivalent of chronic light exposure in rod and cone photoreceptors.

[00115) Mutations in Gucy2d account for ~15% of all cases of LCA making it one of the leading causes of this disease. The number of patients affected by LCA1 is approximately double that affected by the well known RPE65 version of the disease (LCA2), a form for which successful AAV-mediated gene therapy trials have recently garnered worldwide attention. Diagnosis of LCA1 is typically made within the first few months of life in an infant with total blindness or severely impaired vision, extinguished electroretinogram (ERG) and pendular nystagmus (Perrault et al., 1999; Chung and Traboulsi, 2009), Despite these functional deficits, LCA1 patients present with normal fundus (Perrault et al., 1999) and retain some rod and cone photoreceptors in both their macular and peripheral retina for years (Milam el al, 2003; Simonelli et al., 2007; Pasadhika et al., 2009). Using spectral-domain optical coherence tomography (SDOCT) to scan the central macular and perifoveal areas, a recent study revealed that LCA1 patients (age range, 20-53 years) retained all 6 retinal layers with visible photoreceptor inner/outer segment juncture. Maintenance of retinal structure in LCA1 is unlike other forms of the disease which exhibit marked retinal thinning that generally worsens with age (Pasadhika etal., 2009). While the preservation of retinal structure does not parallel better visual acuity in LCA I patients, it does suggest that they are better suited for future therapeutic strategies.

Animal Models [00116) Two animal models carrying null mutations in the retGCl gene have been used to evaluate gene replacement therapy, the naturally occurring GUCY1*B chicken and the guanylate-cyclase-1 (GC1) knockout mouse (see e.g., Williams et al, 2006; Haire et al, 2006). The GUCY1*B chicken is blind at hatch, exhibits extinguished scotopic (rodmediated) and photopic (cone-mediated) ERG and retinal degeneration (see e.g., Ulshafer et

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2018203034 01 May 2018 al., 1984; Huang et al., 1998; Semple-Rowland et al„ 1998). Lentiviral-mediated transfer of Gucy2d to the GUCY1 *B retina restored vision to these animals as evidenced by behavioral testing and ERG (see e.g., Williams et al., 2006). Despite the short term therapeutic success, this therapy fell short of preserving retinal structure or function in the long term. The transient nature of this result, obtained in a non-mammalian species with an integrating viral vector delivered in ovo suggested the need for more appropriate translational studies towards the development of clinical application.

[00117( A mammalian model of LCA1, the GC1 KO mouse, exhibits cone photoreceptor degeneration (see e.g., Yang et al., 1999; Coleman et al., 2004). Like LCA1 patients, loss of cone function in this mouse model precedes cone degeneration (Yang et al., 3999), In addition, light-induced translocation of cone arrestin is disrupted. Rod photoreceptors in this model do not degenerate and continue to generate electrical responses to light (Yang et al.,

1999), a result likely owed to the presence of GC2, a close relative of GC1 in these cells (see e.g., Lowe et al., 1995; Yang et at., 1995; Yang and Garbers, 1997; Karan et al., 2010). AAV-mediated transfer of Gucy2d to the post-natal GC1KO retina restored light-driven translocation of cone arrestin in transduced cells, but failed to restore cone ERG responses or prevent cone degeneration (Haire et al., 2006). In both the chicken and mouse studies, which were conducted by the same investigators, the therapeutic cDNA was of bovine origin which is the protein species historically used in biochemical assays evaluating GC1 functionality (Otto-Bruc etal., 1997; Williams etal., 2006).

Exemplary Definitions (00118] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and compositions similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods

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2018203034 01 May 2018 and compositions are described herein. For purposes of the present invention, the following terms are defined below:

(00119] In accordance with long standing patent law convention, the words “a” and “an” when used in this application, including the claims, denotes “one or more.” [00120] As used herein, the term “about” should generally be understood to refer to both numbers in a range of numerals. For example, “about 1 to 10” should be understood as “about 1 to about 10.” Moreover, all numerical ranges herein should be understood to include each whole integer within the range, as well as each tenth. The term “about,” as used herein, should generally be understood to mean “approximately”, and typically refers to numbers approximately equal to a given number recited within a range of numerals. Moreover, all numerical ranges herein should be understood to include each whole integer within the range. |0012I] In accordance with the present invention, polynucleotides, nucleic acid segments, nucleic acid sequences, and the like, include, but are not limited to, DNAs (including and not limited to genomic or extragenomic DNAs), genes, peptide nucleic acids (PNAs) RNAs (including, but not limited to, rRNAs, mRNAs and tRNAs), nucleosides, and suitable nucleic acid segments either obtained from natural sources, chemically synthesized, modified, or otherwise prepared or synthesized in whole or in part by the hand of man.

[00122] As used herein, the term “nucleic acid” includes one or more types of; polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing Dribose), and any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases (including abasic sites). The term “nucleic acid,” as used herein, also includes polymers of ribonucleosides or deoxyribonucleosides that are covalently bonded, typically by phosphodiester linkages between subunits, but in some cases by phosphorothioates, methylphosphonates, and the like. “Nucleic acids” include single- and double-stranded DNA, as well as single- and double-stranded RNA. Exemplary nucleic acids include, without limitation, gDNA; hnRNA; mRNA; rRNA, tRNA, micro RNA

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2018203034 01 May 2018 (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snORNA), small nuclear RNA (snRNA), and small temporal RNA (stRNA), and the like, and any combination thereof. [00123] As used herein, the term “DNA segment” refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment obtained from a biological sample using one of the compositions disclosed herein refers to one or more DNA segments that have been isolated away from, or purified free from, total genomic DNA of the particular species from which they are obtained. Included within the term “DNA segment,” are DNA segments and smaller fragments of such segments, as well as recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. [001241 Similarly, the term “RNA segment” refers to an RNA molecule that has been isolated free of total cellular RNA of a particular species. Therefore, RNA segments can refer to one or more RNA segments (either of native or synthetic origin) that have been isolated away from, or purified free from, other RNAs. Included within the term “RNA segment,” are RNA segments and smaller fragments of such segments.

[00125] In the context of the invention the term “expression” is intended to include the combination of intracellular processes, including transcription and translation undergone by a polynucleotide such as a structural gene to synthesize the encoded peptide or polypeptide. 100126) The term “e.g,,” as used herein, is used merely by way of example, without limitation intended, and should not he construed as referring only those items explicitly enumerated in the specification.

[00127J As used herein, the term “promoter” is intended to generally describe the region or regions of a nucleic acid sequence that regulates transcription.

[00128| As used herein, the term “regulatory element” is intended to generally describe the region or regions of a nucleic acid sequence that regulates transcription. Exemplary regulatory elements include, but are not limited to, enhancers, post-transcriptional elements, transcriptional control sequences, and such like.

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2018203034 01 May 2018 [00129] As used herein, the term structural gene” is intended to generally describe a polynucleotide, such as a gene, that is expressed to produce an encoded peptide, polypeptide, protein, ribozyme, catalytic RNA molecule, or antisense molecule.

(00130] As used herein, the term “transformation” is intended to generally describe a process of introducing an exogenous polynucleotide sequence (e.g,, a viral vector, a plasmid, or a recombinant DNA or RNA molecule) into a host cell or protoplast in which the exogenous polynucleotide is incorporated into at least a first chromosome or is capable of autonomous replication within the transformed host cell. Transfection, electroporation, and “naked” nucleic acid uptake all represent examples of techniques used to transform a host cell with one or more polynucleotides.

|00131] As used herein, the term “transformed cell” is intended to mean a host cell whose nucleic acid complement has been altered by the introduction of one or more exogenous polynucleotides into that cell, (00132] As used herein, the term “transgenic cell” is generally intended to mean any cell that is derived or regenerated from a transformed cell or derived from another transgenic cell, or from the progeny or offspring of any generation of such a transformed or transgenic host cell. (001331 As used herein, the term “vector” is generally intended to mean a nucleic acid molecule (typically comprised of DNA) capable of replication in a host cell and/or to which another nucleic acid segment can be operatively linked so as to bring about replication of the attached segment. A plasmid, cosmid, or a virus is an exemplary vector.

{00134] The terms “substantially corresponds to,” “substantially homologous,” or “substantial identity” as used herein denotes a characteristic of a nucleic acid or an amino acid sequence, wherein a selected nucleic acid or amino acid sequence has at least about 70 or about 75 percent sequence identity as compared to a selected reference nucleic acid or amino acid sequence. More typically, the selected sequence and the reference sequence will have at least about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84 or

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2018203034 01 May 2018 even about 85 percent sequence identity, and more preferably at least about 86% sequence identity, at least about 87% sequence identity, at least about 88% sequence identity, at least about 89% sequence identity, at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, or at least about 95% percent or greater sequence identity. More preferably still, highly homologous sequences often share greater than at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity between the selected sequence and the reference sequence to which it was compared. The percentage of sequence identity may be calculated over the entire length of the sequences to be compared, or may be calculated by excluding small deletions or additions which total less than about 25 percent or so of the chosen reference sequence. The reference sequence may be a subset of a larger sequence, such as a portion of a gene or flanking sequence, or a repetitive portion of a chromosome, 100135) However, in the case of sequence homology of two or more polynucleotide sequences, the reference sequence and the target sequence will typically comprise at least about 18 to about 25 contiguous identical nucleotides, more typically at least about 26 to about 35 contiguous nucleotides that are identical, and even more typically at least about 40, about 50, about 60, about 70, about 80, about 90, or even about 100 or so contiguous nucleotides that are identical. Desirably, which highly homologous fragments are desired, the extent of overall percent sequence identity between two given sequences will be at least about 80% identical preferably at least about 85% identical, and more preferably about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, or even about 95% or greater identical, as readily determined by one or more of the sequence comparison algorithms well-known to those of skill in the art, such as, e.g., the FASTA program analysis described by Pearson and Lipman (1988).

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2018203034 01 May 2018 [00136] The terms “identical” or percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (or other algorithms available to persons of ordinary skill) or by visual inspection.

[001371 The phrase “substantially identical,” in the context of two nucleic acids refers to two or more sequences or subsequences that have at least about 90%, preferably 91%, most preferably about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 98.5%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about

99.5%, about 99.6%, about 99.7%, about 99.8%i, or about 99.9% or more nucleotide residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. Such “substantially identical” sequences are typically considered “homologous, without reference to actual ancestry.

[00138] The term “naturally occurring” as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by the hand of man in a laboratory is naturallyoccurring. As used herein, laboratory strains of rodents that may have been selectively bred according to classical genetics are considered naturally occurring animals.

[00139[ As used herein, a “heterologous” is defined in relation to a predetermined referenced gene sequence. For example, with respect to a structural gene sequence, a heterologous promoter is defined as a promoter which does not naturally occur adjacent to the referenced structural gene, but which is positioned by laboratory manipulation. Likewise, a heterologous gene or nucleic acid segment is defined as a gene or segment that does not naturally occur adjacent to the referenced promoter and/or enhancer elements.

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100140] As used herein, the term “homology” refers to a degree of complementarity between two or more polynucleotide or polypeptide sequences. The word “identity” may substitute for the word “homology” when a first nucleic acid or amino acid sequence has the exact same primary sequence as a second nucleic acid or amino acid sequence. Sequence homology and sequence identity can be determined by analyzing two or more sequences using algorithms and computer programs known in the art. Such methods may be used to assess whether a given sequence is identical or homologous to another selected sequence.

100141] As used herein, “homologous” means, when referring to polynucleotides, sequences that have the same essential nucleotide sequence, despite arising from different origins. Typically, homologous nucleic acid sequences are derived from closely related genes or organisms possessing one or more substantially similar genomic sequences. By contrast, an “analogous” polynucleotide is one that shares the same function with a polynucleotide from a different species or organism, but may have a significantly different primary nucleotide sequence that encodes one or more proteins or polypeptides that accomplish similar functions or possess similar biological activity. Analogous polynucleotides may often be derived from two or more organisms that are not closely related (e.g., either genetically or phylogenetically).

100142} As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and includes any chain or chains of two or more amino acids. Thus, as used herein, terms including, but not limited to “peptide,” “dipeptide,” “tripeptide,” “protein,” “enzyme,” “amino acid chain,” and “contiguous amino acid sequence” are all encompassed within the definition of a “polypeptide, and the term “polypeptide” can be used instead of, or interchangeably with, any of these terms. The term further includes polypeptides that have undergone one or more post-translational modification(s), including for example, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, post-translation processing,

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2018203034 01 May 2018 or modification by inclusion of one or more non-naturally occurring amino acids. Conventional nomenclature exists in the art for polynucleotide and polypeptide structures. For example, one-letter and three-letter abbreviations are widely employed to describe amino acids: Alanine (A; Ala), Arginine (R; Arg), Asparagine (N; Asn), Aspartic Acid (D; Asp), Cysteine (C; Cys), Glutamine (Q; Gin), Glutamic Acid (E; Glu), Glycine (G; Gly), Histidine (H; His), Isoleucine (I; Ile), Leucine (L; Leu), Methionine (M; Met), Phenylalanine (F; Phe), Proline (P; Pro), Serine (S; Ser), Threonine (T; Thr), Tryptophan (W; Trp), Tyrosine (Y; Tyr), Valine (V; Val), and Lysine (K; Lys). Amino acid residues described herein are preferred to be in the “l” isomeric form. However, residues in the “D” isomeric form may be substituted for any L-amino acid residue provided the desired properties of the polypeptide are retained. [00143] “Protein” is used herein interchangeably with “peptide” and “polypeptide,” and includes both peptides and polypeptides produced synthetically, recombinantly, or in vitro and peptides and polypeptides expressed in vivo after nucleic acid sequences are administered into a host animal or human subject. The term “polypeptide” is preferably intended to refer to all amino acid chain lengths, including those of short peptides of about 2 to about 20 amino acid residues in length, oligopeptides of about 10 to about 100 amino acid residues in length, and polypeptides of about 100 to about 5,000 or more amino acid residues in length. The term “sequence,” when referring to amino acids, relates to all or a portion of the linear N-terminal to C-terminal order of amino acids within a given amino acid chain, e.g., polypeptide or protein; subsequence means any consecutive stretch of amino acids within a sequence, e.g., at least 3 consecutive amino acids within a given protein or polypeptide sequence. With reference to nucleotide and polynucleotide chains, “sequence” and “subsequence” have similar meanings relating to the 5' to 3' order of nucleotides.

]00144| As used herein, the term “substantially homologous” encompasses two or more biomolecular sequences that are significantly similar to each other at the primary nucleotide sequence level. For example, in the context of two or more nucleic acid sequences,

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2018203034 01 May 2018 “substantially homologous” can refer to at least about 75%, preferably at least about 80%, and more preferably at least about 85%, or at least about 90% identity, and even more preferably at least about 95%, more preferably at least about 97% identical, more preferably at least about 98% identical, more preferably at least about 99% identical, and even more preferably still, entirely identical (fe., 100% or “invariant”).

[00145] Likewise, as used herein, the term “substantially identical” encompasses two or more biomolecular sequences (and in particular polynucleotide sequences) that exhibit a high degree of identity to each other at the nucleotide level. For example, in the context of two or more nucleic acid sequences, “substantially identical” can refer to sequences that at least about 80%, and more preferably at least about 85% or at least about 90% identical to each other, and even more preferably at least about 95%, more preferably at least about 97% identical, more preferably at least about 98% identical, more preferably at least about 99% identical, and even more preferably still, entirely identical (i.e., 100% identical or “nondegenerate”).

(00146] The term “recombinant” indicates that the material (e.g., a polynucleotide or a polypeptide) has been artificially or synthetically (non-naturally) altered by human intervention. The alteration can be performed on the material within or removed from, its natural environment or state. Specifically, e.g., a promoter sequence is “recombinant” when it is produced by the expression of a nucleic acid segment engineered by the hand of man. For example, a “recombinant nucleic acid” is one that is made by recombining nucleic acids, e.g., during cloning, DNA shuffling or other procedures, or by chemical or other mutagenesis; a “recombinant polypeptide” or “recombinant protein” is a polypeptide or protein which is produced by expression of a recombinant nucleic acid; and a “recombinant virus,” e.g., a recombinant AAV virus, is produced by the expression of a recombinant nucleic acid, |00147] As used herein, the term “operably linked” refers to a linkage of two or more polynucleotides or two or more nucleic acid sequences in a functional relationship, A nucleic

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2018203034 01 May 2018 acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. “Operably linked” means that the nucleic acid sequences being linked are typically contiguous, or substantially contiguous, and, where necessary to join two protein coding regions, contiguous and in reading frame. Since enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths; however, some polynucleotide elements may be operably linked but not contiguous.

[00148] “Transcriptional regulatory element” refers to a polynucleotide sequence that activates transcription alone or in combination with one or more other nucleic acid sequences. A transcriptional regulatory element can, for example, comprise one or more promoters, one or more response elements, one or more negative regulatory elements, and/or one or more enhancers.

[00149[ As used herein, a “transcription factor recognition site” and a transcription factor binding site” refer to a polynucleotide sequencefs) or sequence motiffs) which are identified as being sites for the sequence-specific interaction of one or more transcription factors, frequently taking the form of direct protein-DNA binding. Typically, transcription factor binding sites can be identified by DNA footprinting, gei mobility shift assays, and the like, and/or can be predicted on the basis of known consensus sequence motifs, or by other methods known to those of skill in the art, [00150| “Transcriptional unit” refers to a polynucleotide sequence that comprises at least a first structural gene operably linked to at least a first cis-acting promoter sequence and optionally linked operably to one or more other cis-acting nucleic acid sequences necessary for efficient transcription of the structural gene sequences, and at least a first distal regulatory element as may be required for the appropriate tissue-specific and developmental transcription of the structural gene sequence operably positioned under the control of the promoter and/or

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2018203034 01 May 2018 enhancer elements, as well as any additional cis sequences that are necessary for efficient transcription and translation (e.g., polyadenylatton site(s), mRNA stability controlling sequence(s), etc.

{00151] The term “substantially complementary,” when used to define either amino acid or nucleic acid sequences, means that a particular subject sequence, for example, an oligonucleotide sequence, is substantially complementary to all or a portion of the selected sequence, and thus will specifically bind to a portion of an mRNA encoding the selected sequence. As such, typically the sequences will be highly complementary to the mRNA target” sequence, and will have no more than about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 or so base mismatches throughout the complementary portion of the sequence. In many instances, it may be desirable for the sequences to be exact matches, i.e., be completely complementary to the sequence to which the oligonucleotide specifically binds, and therefore have zero mismatches along the complementary stretch. As such, highly complementary sequences will typically bind quite specifically to the target sequence region of the mRNA and will therefore be highly efficient in reducing, and/or even inhibiting the translation of the target mRNA sequence into polypeptide product.

[001521 Substantially complementary nucleic acid sequences will be greater than about 80 percent complementary (or “% exact-match) to a corresponding nucleic acid target sequence to which the nucleic acid specifically binds, and will, more preferably be greater than about 85 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds. In certain aspects, as described above, it will be desirable to have even more substantially complementary nucleic acid sequences for use in the practice of the invention, and in such instances, the nucleic acid sequences will be greater than about 90 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds, and may in certain embodiments be greater than about 95 percent

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2018203034 01 May 2018 complementary to the corresponding target sequence to which the nucleic acid specifically binds, and even up to and including about 96%, about 97%, about 98%, about 99%, and even about 100% exact match complementary to all or a portion of the target sequence to which the designed nucleic acid specifically binds,

J00153] Percent similarity or percent complementary of any of the disclosed nucleic acid sequences may be determined, for example, by comparing sequence information using the GAP computer program, version 6,0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (1970). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) that are similar, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess (1986), (2) a penalty of 3,0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

[00154] As used herein, the terms “protein,” “polypeptide,” and “peptide” are used interchangeably, and include molecules that include at least one amide bond linking two or more amino acid residues together. Although used interchangeably, in general, a peptide is a relatively short (e.g., from 2 to about 100 amino acid residues in length) molecule, while a protein or a polypeptide is a relatively longer polymer (eg·., 100 or more residues in length). However, unless specifically defined by a chain length, the terms peptide, polypeptide, and protein are used interchangeably.

[00155) As used herein, the term “patient” (also interchangeably referaed lo as “host” or “subject”) refers lo any host that can serve as a recipient for one or more of the rAAV-based guanylate cyclase compositions as discussed herein. In certain aspects, the recipient will be a vertebrate animal, which is intended to denote any animal species (and preferably, a

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2018203034 01 May 2018 mammalian species such as a human being). In certain embodiments, a “patient” refers to any animal host, including but not limited to, human and non-human primates, bovines, canines, caprines, cavines, corvines, epines, equines, felines, hircines, lapines, leporines, lupines, murines, ovines, porcines, racines, vulpines, and the like, including, without limitation, domesticated livestock, herding or migratory animals, exotics or zoological specimens, as well as companion animals, pets, and any animal under the care of a veterinary practitioner. {00156| As used herein, the term “carrier” is intended to include any solvent(s), dispersion medium, coating(s), diluent(s), buffer(s), isotonic agent(s), solution(s), suspension^), colloid(s), inert(s) or such like, or a combination thereof that is pharmaceutically acceptable for administration to the relevant animal or acceptable for a diagnostic purpose, as applicable. The use of one or more delivery vehicles for gene therapy constructs, viral particles, vectors, and the like, is well known to those of ordinary skill in the pharmaceutical and molecular arts. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the prophylactic, and/or therapeutic compositions is contemplated. One or more supplementary active ingredient(s) may also be incorporated into, or administered in association with, one or more of the disclosed compositions, |00157] As used herein, “an effective amount” would be understood by those of ordinary skill in the art to provide a therapeutic, prophylactic, or otherwise beneficial effect to a recipient patient.

|00158] The phrases “isolated” or “biologically pure” refer to material that is substantially, or essentially, free from components that normally accompany the material as it is found in its native state. Thus, isolated polynucleotides in accordance with the invention preferably do not contain materials normally associated with those polynucleotides in their natural, or in situ, environment.

[00159] “Link or “join” refers to any method known in the art for functionally connecting one or more proteins, peptides, nucleic acids, or polynucleotides, including, without limitation,

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2018203034 01 May 2018 recombinant fusion, covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding, electrostatic bonding, and the like.

(00160] As used herein, the term “plasmid” or “vector” refers to a genetic construct that is composed of genetic material (t,e„ nucleic acids). Typically, a plasmid or a vector contains an origin of replication that is functional in bacterial host cells, e.g., Escherichia coli, and selectable markers for detecting bacterial host cells including die plasmid. Plasmids and vectors of the present invention may include one or more genetic elements as described herein arranged such that an inserted coding sequence can be transcribed and translated in a suitable expression cells. In addition, the plasmid or vector may include one or more nucleic acid segments, genes, promoters, enhancers, activators, multiple cloning regions, or any combination thereof, including segments that are obtained from or derived from one or more natural and/or artificial sources.

[00161] The term “a sequence essentially as set forth in SEQ ID NO:X” means that the sequence substantially corresponds to a portion of SEQIDNO:X and has relatively few nucleotides (or amino acids in the case of polypeptide sequences) that are not identical to, or a biologically functional equivalent of, the nucleotides (or amino acids) of SEQ ID NO:X. The term “biologically functional equivalent” is well understood in the art, and is further defined in detail herein. Accordingly, sequences that have about 85% to about 90%; or more preferably, about 91% to about 95%; or even more preferably, about 96% to about 99%; of nucleotides that are identical or functionally equivalent to one or more of the nucleotide sequences provided herein are particularly contemplated to be useful in the practice of the invention.

[00162] Suitable standard hybridization conditions for the present invention include, for example, hybridization in 50% formamide, 5x Denhardts' solution, 5x SSC, 25 mM sodium phosphate, 0.1% SDS and 100 qg/ml of denatured salmon sperm DNA at 42°C for 16 h followed by 1 hr sequential washes with 0.1 x SSC, 0.1 % SDS solution at 60°C to remove the

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2018203034 01 May 2018 desired amount of background signal. Lower stringency hybridization conditions for the present invention include, for example, hybridization in 35% formamide, 5x Denhardts' solution, 5x SSC, 25 mM sodium phosphate, 0.1% SDS and 100 pg/ml denatured salmon sperm DNA or E. coli DNA at 42°C for 16 h followed by sequential washes with 0.8x SSC, 0.1% SDS at 55°C. Those of skill in the art will recognize that conditions can be readily adjusted to obtain the desired level of stringency.

100163] Naturally, the present invention also encompasses nucleic acid segments that are complementary, essentially complementary, and/or substantially complementary to at least one or more of the specific nucleotide sequences specifically set forth herein. Nucleic acid sequences that are “complementary” are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules. As used herein, the term “complementary sequences” means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to one or more of the specific nucleic acid segments disclosed herein under relatively stringent conditions such as those described immediately above,

100164} As described above, the probes and primers of the present invention may be of any length. By assigning numeric values to a sequence, for example, the first residue is 1, the second residue is 2, etc., an algorithm defining all probes or primers contained within a given sequence can be proposed:

100165} n to n + y, where n is an integer from 1 to the last number of the sequence and y is the length of the probe or primer minus one, where n + y does not exceed the last number of the sequence. Thus, for a 25-basepair probe or primer (i.e., a “25-mer”), the collection of probes or primers correspond to bases 1 to 25, bases 2 to 26, bases 3 to 27, bases 4 to 28, and so on over the entire length of the sequence. Similarly, for a 35-basepair probe or primer (i.e., a “35-mer), exemplary primer or probe sequence include, without limitation, sequences corresponding to bases 1 to 35, bases 2 to 36, bases 3 to 37, bases 4 to 38, and so on over the

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2018203034 01 May 2018 entire length of the sequence. Likewise, for 40-mers, such probes or primers may correspond to the nucleotides from the first basepair to bp 40, from the second bp of the sequence to bp 41, from the third bp to bp 42, and so forth, while for 50-mers, such probes or primers may correspond to a nucleotide sequence extending from bp 1 to bp 50, from bp 2 to bp 51, from bp 3 to bp 52, from bp 4 to bp 53, and so forth, )00166] In certain embodiments, it will be advantageous to employ one or more nucleic acid segments of the present invention in combination with an appropriate detectable marker (i.e., a “label,”), such as in the case of employing labeled polynucleotide probes in determining the presence of a given target sequence in a hybridization assay, A wide variety of appropriate indicator compounds and compositions are known in the art for labeling oligonucleotide probes, including, without limitation, fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, etc., which are capable of being detected in a suitable assay, in particular embodiments, one may also employ one or more fluorescent labels or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally less-desirable reagents. In the case of enzyme tags, colorimetric, chromogenic, or fluorigenic indicator substrates are known that can be employed to provide a method for detecting the sample that is visible to the human eye, or by analytical methods such as scintigraphy, fluorimetry, spectrophotometry, and the like, to identify specific hybridization with samples containing one or more complementary or substantially complementary nucleic acid sequences. In the case of so-called “multiplexing” assays, where two or more labeled probes are detected either simultaneously or sequentially, it may be desirable to label a first oligonucleotide probe with a first label having a first detection property or parameter (for example, an emission and/or excitation spectral maximum), which also labeled a second oligonucleotide probe with a second label having a second detection property or parameter that is different (i.e., discreet or discernable from the first label. The

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2018203034 01 May 2018 use of multiplexing assays, particularly in the context of genetic amplification/detection protocols are well-known to those of ordinary skill in the molecular genetic aits.

Examples [001671 The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 - AAV-Mediated Gene Therapy Restores Visual Function and Behavior to a Mouse Model of LCA1 [00168] In this example, the inventors evaluated whether delivery of a species-specific version of retGCl (i.e., murine) to cone cells of the postnatal GC1KO mouse could restore function to these cells. Serotype 5 AAV vectors were used to deliver mGCl to photoreceptors of postnatal day 14 (Pl4) GC1KO mice. Electroretinogram (ERG) and behavioral testing were used to assess visual function and immunocytochemistry was used to examine therapeutic transgene expression, cone arrestin localization and cone photoreceptor densities in treated and untreated eyes.

[001691 This example demonstrates that an AAV vector subretinally delivered to one eye of P14 GC1 KO mice facilitated expression of wild type retGCl, restoration of visual function and behavior, and preservation of cone photoreceptors. Four weeks following injection,

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2018203034 01 May 2018 visual function (ERG) was analyzed in treated and untreated eyes. ERG was performed every two weeks thereafter until 3 months post injection (the latest time point evaluated). Mice with positive ERG responses as well as isogenic wild type and un-injected control mice were evaluated for restoration of visual behavior using optokinetic reflex testing. At 3 months post injection, all animals were sacrificed and their treated and untreated retinas were evaluated for expression of GC1 and localization of cone arrestin.

(001701 The results also confirm that cone-mediated function was restored to treated eyes of GC1 KO mice (ERG amplitudes were -60% of normal). Moreover, the treatment effect was stable for at least 3 months post- administration. Behavior testing revealed robust improvements in cone-mediated visual behavior, with responses of treated mice being similar or identical to that of wild type mice. Histology revealed AAV-mediated GC1 expression in photoreceptors and a restoration of cone arrestin translocation in treated mice. In addition, cone cell densities were higher in treated eyes than untreated contralateral controls. This result suggests that treatment is capable of preserving cone photoreceptors for at least three months post treatment. This is the first demonstration that postnatal gene therapy is capable of restoring visual function and behavior to, and preserving retinal structure in, a mammalian model of LCA1, Importantly, results were obtained using a well characterized, clinically relevant AAV vector; the in vivo animal model data thus obtained provide the foundation for an AA V-based gene therapy vector for treatment of children affected with LC A1.

Materials and Methods:

Experimental Animals;

(00171( GC1 +/- heterozygote embryos were removed from a cryopreserved stock at The Jackson Laboratory (Bar Harbor, ME, USA). Heterozygotes were mated at the inventors’ facilities to produce GC1 KO (-/-) and isogenic +/+ control offspring. All mice were bred and maintained in a centralized facility at the inventors’ institution under a 12hr/I2hr light/dark

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2018203034 01 May 2018 cycle. Food and water were available ad libitum. All animal studies were approved by the local Institutional Animal Care and Use Committee and conducted in accordance with the

ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and NIH regulations.

Construction of AA V Vectors;

[001721 Serotype 5 Adeno-associated virus (AAV5) vectors were used to deliver murine GC1 (mGCl) as they have been shown to exhibit robust transduction efficiency and a faster onset of expression in retinal photoreceptors than other AAV serotypes (Yang et al., 2002). Both a cell-specific and a ubiquitous promoter were selected to drive expression of mGC 1. The cellspecific, G protein-coupled receptor kinase 1 (GRKJ), also known as rhodopsin kinase promoter was chosen for its ability to specifically target robust transgene expression in rod and cone photoreceptors when used in conjunction with AAV (Khani et at, 2007). The ubiquitous smCBA promoter which exhibits a similar expression pattern to full-length CBA in retina was chosen for its ability to efficiently target the neural retina (Haire el at, 2006). Polymerase chain reaction utilizing the following forward primer:

5-AAAAGCGGCCGCATGAGCGCTTGGCTCCTGCCAGCC-3' (SEQ ID NO: 14) and the following reverse primer;

5'-AAAAGCGGCCGCTCACTTCCCAGTAAACTGGCCTGG-3' (SEQ ID NO: 15) was used to amplify mGC 1 from a plasmid containing a mGC 1 -eGFP fusion (Bhowmick et af. 2009). The resulting fragment was cloned into pCRblunt plasmid (Invitrogen, Carlsbad, CA, USA) and sequence verified. AAV vector plasmid containing smCBA driving expression of mGCl (pTR-smCBA-mGCl) was created by replacing full-length CBA with smCBA in plasmid pTR-CB^SB-hRPE65 (Jacobson et at, 2006) via EcoRl digestion and subsequent ligation. Subsequently, hRPE65 was replaced with mGCl via Notl digestion and ligation, resulting in the creation of pTR-smCBA-mGCl (FIG. 11). An

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AAV vector plasmid containing human GRK1 promoter driving expression of mGCl, pTR-GRKl-mGCl was created by removing hGFP from pTR-hGRKl-hGFP (Beltran et al., 2010) and replacing it with mGCl via Noll digest and ligation (FIG. 11), AAV vectors were packaged according to previously published methods (Haire et al., 2006). Viral particles were resuspended in Balanced Salt Solution (Alcon, Fort Worth, TX, USA) and titered by quantitative real-time PCR (Jacobson et al., 2006). Resulting titers were 4.69 x 10¹² viral genomes per mL (vg/mL) and 4,12 χ I0¹² vg/mL for AAV5-smCBAmGCl and AAV5-hGRKl-mGCl, respectively.

Subretinal Injections:

[00173] One μΐ of AAV5-GRKl-mGCl (4.12 χ 10¹⁰ delivered vector genomes) or AAV5smCBA-mGCl (4.69 χ 10⁹ delivered vector genomes) was delivered subretinally at postnatal day 14 (Pl 4) to the right eye of each GC1 KO mouse, leaving the left eye as a contralateral control. Subretinal injections were performed as previously described (Timmers et al., 2001; Pang et al., 2006). Further analysis was carried out only on animals which received comparable, successful injections (>60% retinal detachment and minimal complications). It is well established that the area of retinal detachment corresponds to the area of viral transduction (Cideciyan et al„ 2008; Timmers et al., 2001).

Electroretinographic Analysis:

[00174) Electroretinograms (ERGs) of treated GC1KO (n=14) and isogenic +/+ controls (n=2) were recorded using a PC-based control and recording unit (Toennies Multiliner Vision; Jaeger/Toennies, Hochberg, Germany) according to methods previously described with minor modifications (Haire et al., 2006). initial ERG measurements were recorded at 4 weeks’ postinjection, and each subsequent 2 weeks thereafter, until 3 months’ post-injection (the latest time point evaluated in the study). Age matched +/+ isogenic controls were recorded

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2018203034 01 May 2018 alongside treated animals at every time point. Mice were dark-adapted overnight (more than 12 hours) and anesthetized with a mixture of 100 mg/kg ketamine, 20 mg/kg xylazine and saline in a 1:1:5 ratio, respectively. Pupils were dilated with 1% tropicamide and 2.5% phenylephrine hydrochloride. A heated circulating water bath was used to maintain the body temperature at 38°C. Hydroxypropyl methylcellulose 2.5% was applied to each eye to prevent corneal dehydration. Full-field ERGs were recorded using custom, gold wire loop corneal electrodes. Reference and ground eiectrodes were placed subcutaneously between the eyes and in the tail, respectively. Scotopic rod recordings were elicited with a series of white flashes of seven increasing intensities (0.01 mcds/m² to 5 cds/m²). Interstimulus intervals for low intensity stimuli were 1,1 second. At the three highest intensities (100 mcds/m², 1 cds/m² and 5 cds/m²), interstimulus intervals were 2.5, 5.0 and 20.0 seconds, respectively. Ten responses were recorded and averaged at each intensity. Mice were then light adapted to a 100 cds/m² white background for 2 min. Photopic cone responses were elicited with a series of five increasing light intensities (100 mcds/m² to 12 cds/m²). Fifty responses were recorded and averaged at each intensity. All stimuli were presented in the presence of the 100 cds/m² background. B-wave amplitudes were defined as the difference between the a-wave troughs to the positive peaks of each waveform.

[00175] Photopic b-wave maximum amplitudes (those generated at 12 cds/m²) of all smCBAmGCl- treated (n = 6) and hGRKl-mGCl- treated (n = 8) GC1KO (both treated and untreated eyes) and isogenic +/+ control mice were averaged and used to generate standard errors. These calculations were made at every time point (4 weeks’-l 3 weeks’ post-injection). This data was imported into Sigma Plot for final graphical presentation. The paired /-test was used to calculate P-values between treated and untreated eyes within each promoter group (smCBA or hGRK.1) and between each promoter group over time (4 weeks post-injection vs. 3 months’ post-injection). The standard /-test was used to calculate P-values between smCBA-mGCl vj. hGRK.1-mGCl treated eyes. Significant difference was defined as a P65

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2018203034 01 May 2018 value <0.05, Because some of the mice from each treated group were temporarily removed from the study for behavioral analyses, the total number of mice averaged and presented at each time point in FIG, 2A and FIG. 2B differs. Three mice from the smCBA-mGCl-treated group were sent for optomotor testing, leaving an “n” of 3 mice used for ERG analysis during the 8, 10 and 12 week measurements (FIG. 2A). Two mice from the hGRKl-mGCl- treated group were sent for optomotor testing, leaving an “n” of 6 used for ERG analysis during the 6, 8, 10 and 12 week measurements (FIG. 2B). All mice sent for behavioral analysis were measured at 13 weeks’ post-injection upon their return to the inventors’ laboratories (smCBAmGCl: n = 3, hGRKl-mGCl: π = 2) following completion of behavioral analyses.

Optomotor Testing:

J0O176J Photopic visual acuities and contrast sensitivities of treated and untreated GC1 KO mouse eyes were measured using a two-altemative forced choice paradigm as described previously (see e g., Umino et al, 2008; Alexander et al, 2007), To test the sensitivity of individual eyes from the same animal we took advantage of the fact that mouse vision has minimal binocular overlap and that the left eye is more sensitive to clockwise rotation and the right to counter-clockwise rotation (Douglas et al, 2005). Thus in the inventors’ “randomizeseparate optomotor protocol, each eye’s acuity and contrast sensitivity threshold was determined separately and simultaneously via stepwise functions for correct responses in both the clockwise and counter-clockwise directions. Correct detection of patterns rotating in the clockwise direction was driven primarily by visual signals originating from the left eye and correct responses in the counterclockwise direction were derived from visual signals originating from the right eye. Acuity was defined the highest spatial frequency (100% contrast) yielding a threshold response, and contrast sensitivity was defined as 100 divided by the lowest percent contrast yielding a threshold response. For photopic acuity, the initial stimulus was a 0,200 cycles/degree sinusoidal pattern with a fixed 100% contrast. For

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2018203034 01 May 2018 photopic contrast sensitivity measurements, the initial pattern was presented at 100% contrast, with a fixed spatial frequency of 0.128 cycles/degree. Photopic vision was measured at a mean luminance of 70 cd/m². Visual acuities and contrast sensitivities were measured for both eyes of each mouse four to six times over a period of 1 week. Age matched, isogenic +/+ control animals (Ml, M2) and naive GCI KO mice (M3, M4) are presented along with the smCBA-mGCl-treated (M5, M6, M7) and hGRKl-mGCl-treated mice (M8, M9) in FIG. 3. Cone-mediated ERG amplitudes generated from a 12 cds/m² stimulus of all mice (MI-M9) are presented alongside the behavior results. Unpaired f-tests were carried out on acuity and percent contrast values to determine significance of results.

Tissue Preparation:

[00177] Three months’ post-injection, P14-treated GC1K.O mice and age matched isogenic +/+ controls were dark adapted for 2 hr. Immediately following dark adaptation, mice were sacrificed under dim red light (>650 nm). The limbus of injected and un-injected eyes was marked with a hot needle at the 12:00 position, facilitating orientation. Enucleation was performed under dim red light and eyes were placed immediately in 4% paraformaldehyde. Eyes that were to be used for cryosectioning were prepared according to previously described methods (Haire et al., 2006). Briefly, corneas were removed from each eye, leaving the lens inside the remaining eye cup. A small “V” shaped cut was made into the sclera adjacent to the burned limbus to maintain orientation. After overnight fixation, the lens and vitreous were removed. The remaining retina/RPE-containing eyecup was placed in 30% sucrose in PBS for at least 1 hr at 4°C. Eyecups were then placed in cryostat compound (Tissue Tek OCT 4583; Sakura Finetek, Inc., Torrance, CA, USA) and snap-frozen in a bath of dry ice/ethanol. Eyes were serially sectioned at 10 pm with a cryostat (Microtome HM550; Walldorf, Germany). Eyes that were to be used for whole mount analysis were prepared according to previously described methods (Pang et al., 2010). Orientation was achieved as previously

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2018203034 01 May 2018 mentioned. After overnight fixation, cornea, lens, vitreous and retinal pigment epithelia were removed from each eye without disturbing the retina, A cut was made in the superior (dorsal) portion of the retina adjacent to the original limbus bum to maintain orientation.

Immunohistochemistry and Microscopy:

[00178) Retinal cryosections and whole mounts were washed 3 x in IX PBS. Following these washes, samples were incubated in 0.5% Triton X-100® for 1 hr in the dark at room temperature. Next, samples were blocked in a solution of 1% bovine serum albumin (BSA) in PBS for 1 hr at room temperature. Retinal sections were incubated overnight at 37°C with a rabbit polyclonal GC1 antibody (1:200, sc-50512, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) or rabbit polyclonal cone arrestin antibody (“Lumij” 1:1000; provided by Dr. Cheryl Craft, University of Southern California, Los Angeles, CA, USA) diluted in 0.3% Triton X-100®/l% BSA. Retinal whole mounts were incubated overnight at room temperature with the same cone arrestin antibody, diluted 1:1000 in 0.3% Triton X-100®/l % BSA. Following primary incubation, retinal sections and whole mounts were washed 3X with

IX PBS.

|00l79] Retinal sections were incubated for 1 hr at room temperature with IgG secondary antibodies tagged with either Alexa-594 or Alexa-488 fluorophore (Molecular Probes, Eugene, OR, USA) diluted 1:500 in IX PBS. Following incubation with secondary antibodies, sections and whole mounts were washed with IX PBS. Retinal sections were counterstained with 4',6'-diamino-2-phenylindole (DAPI) for 5 min at room temperature. After a final rinse with IX PBS and water, sections were mounted in an aqueous-based medium (DAKO) and cover-slipped. Retinal whole mounts were oriented on slides with the superior (dorsal) portion of the retina positioned at the 12:00 position. Samples were mounted in DAKO and cover-slipped.

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100180] Retinal sections were analyzed with confocal microscopy (Leica TCS SP2 AOBS Spectral Confocal Microscope equipped with LCS Version 2.61, Build 1537 software, (Bannockburn, IL, USA). AU images were taken with identical exposure settings at either 20x or 63x magnification. Excitation wavelengths used for DAPI, GC1 and cone arrestin stains were 405 nm, 488 nm, and 594 nm, respectively. Emission spectra were 440-470 nm, 500-535 nm and 605-660 nm, respectively. Retinal whole mounts were analyzed with a widefield fluorescent microscope (Axioplan 2) (Zeiss, Thornwood, NY, USA) equipped with a Qlmaging Retiga 4000R Camera and Qlmaging QCapture Pro software (Qlmaging, Inc., Surrey, BC, Canada). Quadrants of each whole mount were imaged at 5x under identical exposure settings and then merged together in Photoshop® (Version 7.0) (Adobe, San Jose, CA, USA)

Image Analysis:

[00181] Cone photoreceptor densities were analyzed in retinal whole mounts by counting cells labelled with secondary fluorophore directed against cone arrestin antibody in the central and inferior retina using ImageJ® software (National Institutes of Health, Bethesda, MD, USA). These values were obtained by zooming in on the 5X TIFF files shown in FIG. 6. Five squares (500 pm²) were placed over identical areas in central and inferior retina of both treated and untreated GC1K.O eyes. For central retina, squares were placed at an equal eccentricity around the optic nerve head in all eyes (125 pm). Cone photoreceptors were counted in each respective retinal area, values were averaged and standard deviations calculated. The standard r-test was used to calculate /’-values between desired samples. Significant difference was defined as a P-value < 0.05.

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Results

Photoreceptor Function (ERG) was restored in AA V-Treated GC1KO Mice: [00182| It was previously reported that cone responses in the GC1KO mouse are barely detectable by 1 month of age. Here the inventors have shown that P14-treatment of this mouse with an AAV vector carrying the mouse GC1 gene under the control of either a photoreceptor-specific (hGRKl) or ubiquitous (smCBA) promoter led to substantial restoration of cone photoreceptor function as measured by ERG. Representative cone traces (FIG. 1) (as well as the average photopic b-wave amplitudes (FIG. 2A and FIG. 2B) from hGRKl-mGCl-treated, smCBA-mGCl-treated, GC1KO and isogenic +/+ controls) showed that cone function in treated eyes was restored to approximately 45% of normal at four weeks’ post-injection. Similar to previous reports, cone responses in contralateral, untreated eyes were ablated by this time point. At 4 weeks’ post-injection, the average cone-mediated b-wave amplitude in smCBA-mGC l -treated eyes (65.1 pV) was significantly higher (^ = 0.006) than that in the untreated eyes (3.9 pV). The average cone mediated b-wave amplitude in hGRKl-mGCl-treated eyes (59.1 pV) was significantly higher (P < 0.001) than that in untreated eyes (3.2 pV). The level of restoration achieved four weeks’ post-delivery of the photoreceptor-specific hGRKl-mGCl vector was not significantly different from that achieved with the ubiquitous promoter-containing smCBA-mGCl vector (^=0.604). At 3 months’ post-injection, the average cone-mediated b-wave amplitude in smCBAmGCl-treated eyes (53.3 pV) was significantly higher (P< 0.001) than that in the untreated eyes (2.8 pV), The average cone mediated b-wave amplitude in hGRKl-mGCl-treated eyes (45.3 pV) was significantly higher (P< 0.001) than that in untreated eyes (3.4 pV). The level of restoration achieved 3 months following delivery of the photoreceptor-specific GRK1mGCl vector was not significantly different from that achieved with the ubiquitous promotercontaining smCBA-mGCl vector (P = 0.331). Both promoters conferred similar levels of functional restoration to cones in treated eyes of the GC1KO mouse in the short term.

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Importantly, restoration of cone photoreceptor function remained stable for 3 months (the latest time point evaluated in this study (see FIG. 1, FIG. 2A and FIG. 2B). There was no significant difference in photoptc b-wave amplitudes of smCBA-mGCl-treated or hGRKlmGCl-treated eyes between 4 weeks and 3 months post treatment (P = 0.174 and 0.125, respectively).

[00183] ERG implicit times which are an important feature in the diagnosis of various retinal disorders including other forms of LCA (Sun et al., 2010) were also determined. While no such measurement can be obtained from a GC1K.0 eye (there are no ERG responses in these eyes), it was possible to compare cone b-wave implicit times in AAV-mGCl treated and isogenic +/+ control mice. At 4 weeks post injection, there was no significant difference between cone b-wave implicit times in treated and +/+ control eyes (AO. 884); average values in AAV-mGCl-treated and +/+ eyes at this time point were 50.8 ms and 50.4 ms, respectively. At 3 months post injection, there was also no significant difference between the two groups (P= 0.697); averages of all cone b-wave implicit times in treated and +/+ control eyes were 59.7 ms and 58.3 ms, respectively. The response kinetics of cones in the treated GC1 KO retina (as determined by implicit time measurements) appeared to be normal and stable in the short term.

[00184] It was previously reported that rod ERGs in the GC1KO mouse show alterations by 1 month of age, with the rod a-wave and b-wave both markedly reduced (Yang et al., 1999). This reduction plateaus at 5 months of age with responses approximately 50-70% that of a wild-type (WT) mouse. While some instances of AAV-mGCl-mediated improvements were observed in treated eyes of GC1KO mice relative to untreated controls (example seen in FIG, 1), this result was not as consistent as that seen in the cone-mediated responses.

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Visual Behavior was Restored in AA V-Treated GCIKO Mice:

100185] Optomotor analysis revealed that eyes of GC1KO mice treated with either smCBAmGCl (M5, M6, M7) or hGRKl-mGCl (M8, M9) responded significantly better than untreated eyes under all photopic, cone-mediated conditions. Untreated GCIKO eyes perform poorly with a visual acuity of 0.163 ± 0,040 cycles per degree (FIG. 3B and FIG. 3C, bar, mean + s.d., n = 9 eyes). Isogenic GC1^+/+ control eyes (Ml, M2) respond significantly better, showing an average acuity of 0,418 ± 0.046 cycles per degree (n = 4 eyes). AAVmGCI-treated eyes (M5-M9) have an average acuity of 0.392 ±0.077 cycles per degree (« = 5 eyes), a level essentially identical to control +/+ eyes and significantly better than untreated GCIKO eyes (P< 0,0001), Photopic contrast sensitivities (FIG, 3B and FIG. 3C) paralleled the photopic acuity results, with AAV-mGCl-treated eyes (contrast sensitivity of 11,9 + 7.37, n = 5 eyes) showing contrast thresholds nearly identical to +/+ mice (11.94+ 3.03, « = 4 eyes). Again, GCIKO eyes treated at P14 with AAV-mGCl performed significantly better than untreated eyes, which showed an average contrast sensitivity of 1,27 + 0.31 (n = 9, P< 0.0001). In all photopic tests, untreated GCIKO eyes perform extremely poorly, essentially equivalent to no cone-mediated function. Statistical comparisons of these measurements are shown in Table 1. Cone-mediated ERG traces of all GCf'⁺ (Ml, M2), GCIKO (M3, M4), smCBA-mGCl-treated (M5, M5, M7) and hGRK.1mGCl-treated (M8, M9) mice used in behavior analysis are shown in FIG, 3A to relate visual function (optmoior behavior) to retinal function (electrophysiology).

100186] Rod retinal function (ERG) is partially preserved in the GCIKO mouse. Studies have shown that even very small ERG amplitudes translate into robust visual behavior (Williams et al., 2006). In fact, LCA2 patients who received AAV-RPE65 therapy were found to exhibit behavioral restoration despite a complete lack of ERG response (Maguire et al., 2008). Optomotor testing revealed that scotopic, rod-mediated visual acuities and contrast sensitivities of GCIKO eyes are very similar to +/+ controls. For this reason, it was

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Statistical comparisons of these measurements are shown in Table 1;

Table 1

Statistical Comparison of the Photopic Visual Functions of WT, AAV-mGCITreated and Untreated GCIKO Eyes as Measured by Optomotor Behavior

Photopic Acuity	Wild Type(WT)	Treated	Untreated
Number of Values Mean Standard Deviation	4 0.4183 0.0456	5 0.3919 0.07731	9 0.163 0.03954
	WT vj. Treated WT vj. Untreated Treated vj. Untreated	P-value 0.5671 <0.0001 <0.0001	Not significant * *
Photopic Contrast Sensitivity	WT	Treated	Untreated
Number of Values Mean Standard Deviation	4 11.94 3.03	5 11.16 7.37	9 1.27 0.31
	WT vj. Treated WT vj. Untreated Treated vj. Untreated	P-value 0.4186 <0.0001 <0.0001	Not significant * *
		*-p< 0.0001

Photoreceptor-Specific and Ubiquitous Promoters Both Drive mGCl

Transgene Expression in Rods and Cones of GCIKO Mice;

[00187] GC1-deficiency affects both rod and cone photoreceptors in LCA1 patients. The photoreceptor-specific human RK promoter and the ubiquitous smCBA promoter were therefore ehosen for this study as a means of targeting both cell types. The human RK promoter was chosen for its small size and ability to efficiently drive transgene expression specifically in photoreceptor cells. Immunostaining of GCIKO retinas 3 months’ posttreatment with AAV-hGRKl-mGCl revealed that this promoter drove robust GC1 expression

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2018203034 01 May 2018 in photoreceptor outer segments. A representative image of a retinal cross section from an eye injected with this therapeutic vector (FIG. 4A) shows intense GCI staining in the OS layer whereas the contralateral, untreated eye from the same mouse lacks any GCI expression (FIG. 4B). The smCBA promoter also efficiently drove GCI expression in photoreceptor cells. Photoreceptor OS exhibited robust smCBA-mediated GCI expression in treated eyes (FIG. 4C), relative to the contralateral, untreated eye (FIG, 4D). Levels of hGRKl and smCBA-mediated GCI expression approached that seen in isogenic, +/+ control eyes (FIG. 4E). GCI expression in hGRKl-mGCl-treated eyes was restricted to OS. In smCBAmGCl-treated eyes, GCI expression was occasionally found in photoreceptor cell bodies of the outer nuclear layer (see e.g., arrows FIG. 4F). Notably however, neither promoter construct drove therapeutic GCI expression outside the photoreceptor cells. This lack of offtarget expression is relevant to the development of future clinical applications.

Cone Arrestin Translocation was Restored in AAV-mGCl-Treated GCJKO

Mice:

J00188] AAV-mGCI treatment restored light-induced cone arrestin translocation to cone photoreceptors in the treated GCI KO retina. Representative treated, untreated and +/+ retinal cross sections immunostained with an antibody generated against cone arrestin showed that cone arrestin was localized to the outer segments, inner segments, axons and synaptic termini of +/+ , smCBA-mGCl-treated and hGRKl-mGCl-treated cone photoreceptors (FIG. 5A, FIG. 5C, and FIG. 5D, respectively). On the contrary, cone arrestin remained localized mostly to the outer segments of cones in untreated GC1KO retinas (FIG. 5B). This result was consistent with the notion that cones in the GC 1 KO mouse retina are chronically hyperpolarized. Not only was a restoration of cone arrestin localization in dark-adapted, treated retinas observed, but an apparent up-regulation of the protein was seen in treated eyes

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2018203034 01 May 2018 relative to untreated controls. Significantly, cone cell densities also appeared higher in treated eyes relative to untreated controls (see e.g., FIG. 5A, FIG. 5B, and FIG, 5C),

Cone Photoreceptors were Preserved in AAV-mGCl-Treated GC1KO Mice: [00189] Analysis of smCBA-mGCl and hGRX-mGCl treated and un-injected, contralateral retinal whole mounts 3 months’ post-injection with therapeutic vector that were stained with an antibody directed against cone arrestin revealed that cone photoreceptors were preserved as a result of treatment with the therapeutic vector (FIG. 6). Counts of cone photoreceptors in inferior and central retinas of both treated and untreated retinal whole mounts revealed that there was a statistically significant difference in the cone cell densities of treated vs. untreated eyes. This result was consistent with the observation that robust electrophysiological and behavioral restoration was clearly evident. P14-treatment of GC1K.O mice with either therapeutic construct was capable of preserving cone photoreceptor structure for at least three months.

Example 2 - animal Model Containing a GC1/GC2 Double Knockout [00190( It is important to note that while only cone photoreceptors are affected in the GC1KO mouse (rods only lose partial function and they do not degenerate), LCA1 patients exhibit rod function loss and rod degeneration. The reason for this difference is speculated to be a speeies-specific difference in the dependence on GC2, a close relative of GC1 that is expressed in rod photoreceptors. Mouse rods are able to function in the absence of GC1 presumably because GC2 is capable of reconstituting activity; however in humans this is not the case. GC1 is required for rod function, hence the rod degeneration. A GC1/GC2 double knockout mouse model was generated and shown to have rod function loss (in addition to cone function loss as seen in the GC1 K/O) (Baehr et al., 2007), It was proven through biochemical studies with this model that GC2 is what provides rod function in the absence of

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GC1. Having said that, it is the GC1/GC2 double knockout mouse that more reliably mimics the human condition (both cones and rods affected) (Karan et al., 2010), To test both the rAAV-smCBA-mGCl and rAAV-hGRKl-mGCl vectors in the GC1/GC2 double knock-out mouse, rAAV vectors are delivered in precisely the same manner and time (post-natal day 14) as with the aforementioned GC1 knock-out study. Analysis of restoration of vision, both physiologically and behaviorally, is also performed in the same manner as described above for the GC1 knock-out study. Particular emphasis is paid to scotopic (Le., rod) responses, as a measurable recovery of rod function is expected in GC1/GC2 double knock-out mice treated with a GO vector construct.

Example 3 - ‘Humanized’ murine Animal Model of LCA1

100191J This example describes the creation of a “humanized murine animal model of LCA1. In one embodiment, the mouse model contains a GC1/GC2/GCAP1 knockout. GCAP1 is the protein that activates GC1. To create an in-vivo system in which human GC1 expressed from a clinical grade rAAV vector designed for use in humans can be evaluated for function, a GC1/GC2/GCAP1 triple knockout hGCAPl transgenic mouse is utilized. In this mouse, visual function is resorted by rAAV-mediated hGCI interacting with hGCAPl only (i.e., no endogenous murine GCAP1 is present). From this study, it is possible to determine whether the human GCAP1 protein is required to stimulate human GC1 activity in the mouse model, and whether function can be restored to cones and rods when the two human polypeptides are reconstituted and expressed in the non-human (i.e., murine) model of the disease. To generate the GC1/GC2/GCAP1 triple knockout hGCAPl transgenic mouse the GC1/GC2 double knockout mouse (Baehr et al, 2007) is crossed with the GCAP1 knock-out mouse (Mendez et al, 2001), Human GCAP1 is then trangenically-expressed in the animal model to generate a GC1/GC2/GCAP1 triple knockout hGCAPl transgenic mouse. Studies in which rAAVvectored hGCI is provided to these animals are conducted in a manner substantially identical

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Example 4 - Exemplary Vector Constructs Useful in the Practice of the

Invention (001921 Maps ^Qf the two illustrative vectors are shown in FIG. 11. One contains the nonspecific promoter smCBA and the other has the rod/cone limited promoter GRK1. Both have been packaged into serotype 5 AAV. Al! vector doses tested to date are safe in the mouse retina. Cohorts of GCI-/- mice were then sub-retinally injected at postnatal day 14 (P14) and then analyzed periodically by ERG and by photopic optokinetic (cone mediated) behavior. Since the GCI' mouse maintains a rod mediated ERG, monitoring functional rescue focused primarily on restoration of cone function. For the smCBA vector ERGs were assessed at 4 weeks post-treatment and every 2 weeks thereafter until 12-13 weeks posttreatment. All 9 eyes treated in 9 mice responded to treatment. The results, shown below demonstrate a significant restoration of photopic ERG amplitudes from essentially unrecordable in control untreated eyes to approximately 50% of normal in partner vector treated eyes.

(001931 Four GCI⁷' mice were then analyzed by scotopic optokinetic behavior for differences mediated by treated vs. untreated partner eyes (shown below). All four treated eyes (289, 290, 294, 295, red bars) showed significant improvement in visual acuity over their control eyes, and three of the four showed significantly improved contrast sensitivity. Mice 297 and 298 were wild type controls, and mouse 299 was an untreated GCI' mouse. The results demonstrate that the vector achieved functional and behavioral restoration of cone mediated vision in the animal model of LC A1,

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J00194] For the GRK.1 vector that limits expression to rods and cones, ERGs were assessed in 14 GC I mice treated in one eye at 4 weeks post-treatment and every 2 weeks thereafter until 12-13 weeks post-treatment. Twelve of the 14 treated eyes in 12 animals responded. The results (shown below) revealed a significant restoration of photopic ERG amplitudes from essentially unrecordable in control untreated eyes to approximately 40% of normal in partner vector treated eyes.

[00195] One GCU mouse (#293) was then analyzed by scotopic optokinetic behavior for differences in treated vs. untreated partner eyes (shown above). This mouse showed significant improvement in both visual acuity and contrast sensitivity in the vector treated right eye (red bar) relative to its control left eye (blue bar). Responses were nearly equivalent to control wild type mice (297 and 298) and significantly improved over an untreated GC1-/mouse (299). It was therefore concluded that the GRK1 vector also achieves functional and behavioral restoration of cone mediated vision in this model of LCA 1.

Example 5 - Specific Cone Targeting of wtGCI Improves Rescue [00196] The data presented above clearly demonstrate that cone function and cone-mediated behavior can be rescued with the rod/cone limited GRK1 promoter. Since human LCA1 shows both rod and cone deficits (unlike the GCl'⁷' mouse that shows primarily cone deficits), expression need not be further limited to gain pure cone specificity. However, there is one final cone phenotype in the mouse model which is important to study: in dark adapted conditions, cone arrestin does not move normally from cone outer segments into inner segments, axons and synaptic termini as it does in the wild type retina. Studies were therefore performed to assess whether this cell biological phenotype was also corrected in vectortreated GC1eyes. In the results shown, a GC1’^A mouse was treated in one eye with the GRK1 vector, then at 7 weeks post-injection the mouse was dark adapted. Treated (bottom panel) and control (top panel) retinas were then analyzed for cone arrestin localization by

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2018203034 01 May 2018 immunohistochemistry. In the untreated GCT retina (top panel), cone arrestin remained largely in cone outer segments (OS) and the synaptic layer (SL). In contrast, in the contralateral treated retina (bottom panel) a substantial fraction (-50%) has translocated into the inner segments and synaptic termini. It was concluded, therefore, that vector treatment also restored correct translocation of cone arrestin.

Example 6 - Long-term Therapy of LCA1 using raaV-vectored Genetic

Constructs [00197] The previous examples have demonstrated that subretinal injection of rAAV vectors containing the murine GC1 cDNA (driven by either the photoreceptor-specific human rhodopsin kinase [hGRKl] or the ubiquitous [smCBA] promoter) were capable of restoring cone-mediated function and visual behavior and preserving cone photoreceptors in the

GC1KO mouse for at least three months.

[00198] In the present Example, the inventors evaluated whether long-term therapy was also achievable in the rodent model of LCA1. Additionally, the inventors examined whether delivery of GC1 to photoreceptors of the GC1/GC2 double knockout mouse (GCdko), a model which exhibits loss of both rod and cone structure and function and phenotypically resembles human LCA1, would confer therapy to these cells.

Methods [00199] Subretinal injections of AAV5-hGRKI-mGCl, AAV5-smCBA-mGCl or the highly efficient capsid tyrosine mutant AAV8(Y733F)-hGRKl-mGCl were performed in one eye of GC1KO or GCdko mice between postnatal day 14 (P14) and P25, Rod and cone photoreceptor function were assayed electroretinographically. Localization of therapeutic GC1 expression and extent of cone photoreceptor preservation were determined by

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2018203034 01 May 2018 immunohistochemistry. Biodistribution studies were used to evaluate the presence of vector genomes in optic nerves and brains of treated animals.

Results |00200] Cone photoreceptor function was restored in GCIKO mice treated with all vectors, with AAV8(733) being the most efficient. Responses were stable for at least 10 months posttreatment. Therapeutic GC1 was found in photoreceptor outer segments. By 10 months postinjection, AAV5 and AAV8(733) vector genomes were detected only in the optic nerves of treated eyes of GCIKO mice. AAV8(733)-vectored mGCl restored function to both rods and cones in treated GCdko mice.

Conclusion (00201] Long-term therapy is achievable in a mammalian model of GC1 deficiency, the GC1 KO mouse, using the rAAV vector constructs disclosed herein. Importantly, therapy was also achievable in the GCdko mouse which mimics the LCA1 rod/cone phenotype. These results provide evidence for die use of rAAV-based gene therapy vectors for treatment of retinal dystrophies, and LCA1 in particular.

Example Ί - Long Term Preservation of Cone Photoreceptors and

Restoration of Cone Function by Gene Therapy in the GCIKO Mouse [00202] In previous examples, it was shown that subretinal AAV5 vectors containing murine GC1 cDNA driven by either the photoreceptor-specific (hGRKl) or the ubiquitous (smCBA) promoter were capable of restoring cone-mediated function and visual behavior and preserving cone photoreceptors in the GCIKO mouse for three months. In the present example, long term therapy is evaluated using the same murine model. AAV5-hGRKlmGCl, AAV5-smCBA-mGCl or the highly efficient capsid tyrosine mutant AAV8(Y733F)86

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2018203034 01 May 2018 hGRKl-mGCl were delivered subretinally to GC1KO mice between postnatal day 14 (P14) and postnatal day (P25). Retinal function was assayed by electroretinograms (ERGs), Localization of AAV-mediated GC1 expression and cone survival were assayed with immunohistochemistry and the spread of vector genomes beyond the retina was quantified by PCR of optic nerve and brain tissue. Cone function was restored with all vectors tested, with AAV8(Y733F) being the most efficient,. AAV-mediated expression of GC1 was found exclusively in photoreceptors. By 10 months post-injection, AAV genomes were detected only in optic nerve of treated eyes. These results demonstrate for the first time that long-term therapy is achievable in a mammalian model of GC1 deficiency.

[00203] Retinal guanyiate cyclase-1 (GC1) encoded by GUCY2D serves a key function in vertebrate phototransduction (Pugh et al., 1997). Following light stimulus, second messenger cyclic GMP (cGMP) is rapidly hydrolyzed by phosphodiesterase (PDE6) within photoreceptor cells leading to a closure of cGMP-gated cation channels and hyperpolarization of the cell. When cytoplasmic [Ca²⁺] drops below 50 nM, GC1 is activated by small Ca²⁺binding proteins, GCAPs (guanyiate cyclase activating proteins). GC1 synthesizes cGMP which binds and reopens cGMP-gated channels, returning the photoreceptor to the dark”, depolarized state (Pugh et al., 1997; Polans et al., 1996; Wensel, 2008; Lamb and Pugh, 2006; Arshavsky et al, 2002), Thus, GC1 plays a vital role in the light-dark and recovery cycles, anchoring, via cGMP, the feedback loop linking intracellular calcium levels and the polarization state of photoreceptors.

[00204J GC1 is expressed in the outer segments of rod and cone photoreceptors of human, monkey and mouse retinas (Dizhoor et al., 1994; Liu et al., 1994; Haire et al., 2006). Like other membrane guanyiate cyclases, it contains an N’-terminal signal sequence, an extracellular domain (BCD), a single transmembrane domain, a kinase-like homology domain (KHD), a dimerization domain (DD) and a C’-temrinal catalytic domain (CCD), and is present likely as homomeric dimers (Yang and Garbers, 1997). Mutations in GUCY2D are

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2018203034 01 May 2018 associated with recessive Leber congenital amaurosis-1 (LCA1) as well as dominant and recessive forms of cone-rod dystrophy, CORD6 and CORD, respectively (PerTault et ai, 1996; Perrault et al., 2000; Kelsell et al., 1998; Perrault et al., 1998; Gregory-Evans et al., 2000; Weigell-Weber et al., 2000; Ugur et al., 2010). LCA1 is a severe, early onset, autosomal recessive blinding disorder characterized by extinguished electroretinogram (ERG) which precedes photoreceptor degeneration (Perrault et al., 1999; Chung and Traboulsi, 2009). CORD6 is a dominant disorder characterized by progressive degeneration of photoreceptors beginning with cones causing early loss of visual acuity and color vision followed by degeneration of rods leading to progressive night blindness and peripheral visual field loss (Kelsell et al., 1998; Perrault et al., 1998). CORD6 mutations are restricted to the dimerization domain (DD) and generally cause an increase in GCAP-mediated activation of GC1 (Payne et al., 2001; Downes et al., 2001; Wilkie et al., 2000). A recently found recessive CORD-causing mutation is located in the catalytic domain (CD) of GC1 and is thought to reduce overall enzyme function (Ugur et al., 2010), LCA1-causing mutations are distributed throughout the ECD, KHD, DD and CCD domains of GC1 (Karan et al., 2010). These mutations alter enzyme structure and stability, may impact retrograde transport of other peripheral membrane associated proteins and are frequently null.

[00205) The GC1KO mouse carries a null mutation in Gucy2e, the murine homologue of GUCY2D. Like LCA1 patients, loss of cone function in this model precedes cone degeneration (Timmers et al., 2001). Rods retain 30-50% of their function and do not degenerate due to the presence of GC2, another functional guanylate cyclase in murine photoreceptors (Yang and Garbers, 1997; Jacobson et al., 2006; Timmers et al., 2001; Cideciyan et al, 2008; Song et al, 2002), In the earlier examples, it was shown that subretinal injection of serotype 5 adeno-associated viral (AAV) vectors containing the murine GC1 cDNA driven by either the photoreceptor-specific human rhodopsin kinase (hGRKl) or the ubiquitous (smCBA) promoter were capable of restoring cone-mediated function and

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2018203034 01 May 2018 visual behavior and preserving cone photoreceptors in the GCI KO mouse for three months. In the present study, AAV-mediated gene replacement therapy was evaluated for its ability to provide therapy to the GCI KO mouse over the long term. AAV5-hGRKl-mGCl and AAV5smCBA-mGCl and the highly efficient capsid tyrosine mutant vector AAV8(Y733F)hGRKl-mGCl were delivered subretinally to GCI KO mice between postnatal day 14 (Pl 4) and postnatal day 25 (P25). These findings demonstrate for the first time that long-term therapy is achievable in a mammalian model of GCI deficiency. Vector genome biodistribution was also evaluated for AAV5- and AAV8(733)-based vectors. These findings have direct bearing on the development of an AAV-based gene therapy clinical trial for LCA1 (and possibly cone-rod dystrophies), and help to develop a standardized vector design for a wide range of recessive retinal degenerations mediated by defects in photoreceptor-associated genes.

Materials and Methods Experimental Animals:

100206] GCI KO and congenic +/+ controls derived from heterozygous matings of GCI+/mice provided by The Jackson Laboratory (Bar Harbor, ME, USA) were bred and maintained in the inventors’ institutional animal care facility under a 12hr/12hr light/dark cycle. Food and water were available ad libitum. All studies were conducted in accordance with the

ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and NIH regulations.

Construction of AA V Vectors:

100207] Serotype 5 Adeno-associated virus (AAV5) vector plasmids containing either the ubiquitous (smCBA) or photoreceptor-specific human rhodopsin kinase (hGRKl) promoter driving murine GCI (mGCl) cDNA were generated according to previously described methods (Boye et al„ 2010). Site-directed mutagenesis of surface-exposed tyrosine residues

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2018203034 01 May 2018 on the AAV2 capsid have been reported (Zhong et al., 2008). Similar methods were used to generate the AAV8(Y733F) capsid mutant described here. All vectors were packaged, purified and titered according to previously described methods (Zolotukhin et al., 2002; Jacobson et al., 2006). Resulting titers for AAV5-smCBA-mGCl, AAV5-hGRKl-mGCl and AAV8(Y733F)-hGRKl-mGCl were 4.69 x 10¹² vector genomes per ml (vg/mL), 4.12 x 10'² vg/mL and 1.08 x 10¹³ vg/mL, respectively.

Subretinal Injections:

[00208] One pL of AAV5-smCBA-mGC 1 (4.69 x KT vector genomes), AAV5-hGRKlmGCl (4.12 x 10¹⁰ vector genomes) or AAV8(Y733F)-hGRKl-mGCl (1.08 x 10^l° vector genomes) were injected subretinally into one eye of GC1KO mice between postnatal day 14 (P14) and and postnatal day 25 (P25). The contralateral control eye remained uninjected, Subretinal injections were performed as previously described (Timmers et al, 2001). Further analysis was carried out only on animals which received comparable, successful injections (>60% retinal detachment with minimal complications). Approximately 75% of all cohorts received “successful” injections. It is well established that the area of vector transduction corresponds to at least the area of retinal detachment (Timmers et al, 2001; Cideciyan et al.,

2008).

Electroretinographic Analysis:

[00209| ERGs of treated GC1KO and age-matched, congenic (+/+) controls were recorded using a PC-based control and recording unit (Toennies Multiliner Vision; Jaeger/Toennies, H6chberg, Germany) according to methods previously described with minor modifications (Haire et al, 2006; Boye et al, 2010). Recordings of AAVS-smCBA-mGCl -treated GC1KO mice (n= 10), AAV5-hGRKl-mGCl-treated GC1KO mice (n = 6), AAV8(Y733F)-treated GC1KO mice (n - 6) and congenic (+/+) controls (n = 8) commenced on different dates and therefore each subset of mice was monitored for slightly different lengths of time, ERGs of

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2018203034 01 May 2018 treated GC1K0 mice were recorded 4-weeks’ post-injection and every month thereafter until 1 year post-injection (AAV5-treated mice) or 9 months post-injection (AAV8[Y73 3 F]-treated mice). Age-matched, congenic (+/+) control mice were followed for 8 months. Mice were removed from the study at different time points throughout the experiment for various postmortem studies (biodistribution studies, retinal immunohistochemical analysis, real time RT-PCR of retina] tissue) or unexpected sickness/death. ERG data was presented only for groups of animals with an n > 3. Therefore, this study compares findings out to 9 months post-injection forAAV5-treated mice and 6 months post-injection for AAV8(Y733F)-treated mice. Treated mice continued to exhibit ERG responses beyond these time points, however sample sizes were sufficiently reduced such that statistical analysis was no longer practical. Representative cone-mediated traces from individual mice 1 year post-treatment with AAV5 vectors and 9 months post-treatment with AAV8(Y733F) are presented to support this contention. Scotopic (rod-mediated) and photopic (cone-mediated) recordings were elicited using recording parameters previously described (Boye et al, 2010). B-wave amplitudes were defined as the difference between the a-wave troughs and the subsequent positive peak of each waveform. Rod-mediated ERG responses in untreated GC1KO mice are variable from animal to animal (Yang et al., 1999), hence, large standard deviations were observed when averaging scotopic a- and b-wave amplitudes from different animals. Rod ERG data is presented in ratio form (the average of intra-individual, treated versus untreated rod a- and bwave amplitudes). As such, any value above 1 indicates AAV-mGCl treatment improved the rod response. Ratios were calculated using amplitudes generated with a 1 cds/m² stimulus. Photopic, cone-mediated b-wave maximum amplitudes in injected and uninjected eyes of all treated GC1K.0 mice and congenic (+/+) control mice generated at 12 cds/m² were averaged at each time point and used to generate standard errors. All data was imported into Sigma Plot for final graphical presentation. The standard /-test was used to calculate P-values between data sets. Significant difference was defined as a P-value <0,05.

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Bindistribution:

[00210| The spread of vector DNA in tissues of the treated GC1KO mice was determined in samples collected at sacrifice according to previously described methods with minor modifications (Jacobson et al., 2006). Vector-treated mice were sacrificed at the following time points: AAV8(Y733F)-hGRKt-mGCl-treated mice (4-months’ post-injection: n = 1; 7months’ post-injection: n= 1), AAV5-smCBA-mGCl (7-months* post-injection: n= 1; 10months’ post-injection: « = 5), AAV5-hGRKl-mGCl (7-months* post injection: n= 1; 10months’ post-injection: n = 1), Control tissues from GC1K0 mice age-matched to the 7month-post injection or 10-month-post injection time points were also evaluated alongside experimental animals. Following sacrifice, different new forceps were used to enucleate treated and untreated eyes which retained approximately 0,5 cm of proximal optic nerve. Different, new dissection scissors were then used to cut the optic nerves away from the eyeballs after which they were snap frozen in liquid nitrogen and transferred to -80°C where they remained until the time of DNA extraction. Eyeballs were immersed in 4% paraformaldehyde (PAF) and processed for immunohistochemistry (see below).

|<H)211] Brains were removed and a stainless steel mouse coronal brain matrix (Harvard Apparatus,Holliston, MA, USA) was used to isolate visual-specific regions. Right and left lateral geniculate nuclei were collected from one mouse per treatment group (at the latest time point), formalin fixed and saved in the event that vector genomes were recovered from brain and immunohistochemistry was necessary. Separate portions of right and left brain containing visual pathways were collected, snap frozen in liquid nitrogen and transferred to -80°C where they remained until the time of DNA extraction. Precautions were taken io avoid cross-contamination while harvesting tissues. Genomic DNA was extracted from tissues according to the manufacturer’s protocol (Qiagen DNeasy tissue kit). Resulting DNA concentrations were determined using an Eppendorf Biophotomoter (Model 6131; Eppendorf,

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Hamburg, Germany). Quantitative PCRs were performed according to previously described methods with minor modifications (Jacobson et at., 2006; Song et at, 2002; Poirier et at, 2004).

[00212} Primer pairs were designed to the SV40 poly-adenyl ation signal (SV40 polyA) region in each vector genome and standard curves established using known concentrations of plasmid DNA containing the same SV40 polyA target sequence. DNA samples were assayed in triplicate. In order to rule out false negatives due to inhibition of PCR, the third replicate was ‘spiked’ with plasmid DNA containing target (SV40 polyA) at a ratio of 100 copies/pg of genomic DNA. If > 40 copies of the spike-in DNA were detected, the sample was considered acceptable for reporting vector genome copies. In some cases samples failing ‘spike in’ were reanalyzed using less than 1 pg of genomic DNA in PCR reactions, thereby diluting out PCR inhibitors copurifying with DNA in the extracted tissue. Spike-in copy number was reduced proportionally to maintain the 100 copies/pg DNA ratio. Criteria for reporting vector genome copies were established according to previously described methods (Jacobson et at, 2006). Briefly, greater than 100 genome copies/pg was considered positive and the measured copy number/pg reported. Fewer than 100 copies/pg was considered negative.

Tissue Preparation, Immunohistochemistry and Microscopy;

(00213) At sacrifice, concomitant with biodistribution studies performed at 7 months post[AAV8(Y733F)-hGRKl-mGCl] and 10 months post- (AAV5-smCBA-mGCI and AAV5hGRKl-mGCl) injection, the limbus of treated GC1KO mice, age-matched, untreated GC1KO mice as well as age-matched congenic GCI+/+ mice were marked with a hot needle at the 12 o’clock position, facilitating orientation. Untreated GCI KO and GCI+/+ controls were age-matched to the AAV8(Y733F)- treated mice (8 months of age at the time of sacrifice). Eyes designated for cryosectioning were processed and immunostained according to previously described methods (Haire et at, 2006), Briefly, 10 pm retinal sections were

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2018203034 01 May 2018 incubated with antibodies directed against GC1 (rabbit polyclonal 1:200, sc-50512 Santa Cruz Biotechnology, USA) or mouse cone arrestin (rabbit polyclonal “LUMIj”, 1:1000, provided by Dr. Cheryl Craft, University of Southern California, Los Angeles, CA, USA). Following primary incubation, IgG secondary antibodies Alexa-488 or Alcxa-594, respectively, were applied for 1 hour at room temperature (1:500 in IX PBS). Sections were counterstained with 4*,6’-diamino-2-phenyl-indole (DAPI) for 5 min at room temperature. At 11-months’ postinjection, one GC1KO mouse that received treatment with AAVS-smCBA-mGCl in one eye only was sacrificed and retinal whole mounts from treated and untreated eyes processed according to previously described methods (Pang et al., 2010). Briefly, whole mounts were stained with LUMIj (1:1000) followed by IgG secondary Alexa-594 (1:500 in IX PBS) and positioned on slides with the superior (dorsal) portion of the retina oriented at 12- o’clock. Retina] sections were analyzed by confocal microscopy (Leica TCS SP2 AOBS Spectral Confocal Microscope equipped with LCS Version 2.61, Build 1537 software). Images were taken at identical exposure settings at 20X magnification. Retinal whole mounts were analyzed with a wide-field fluorescent microscope (Zeiss Axioplan 2) equipped with Qlmaging Retiga 4000R Camera and Qlmaging QCapture Pro software. Quadrants of each whole mount were imaged at 10X under identical exposure settings and then merged together in Adobe Photoshop.

Immunoblotting:

|00214[ At 7 months post-injection, one mouse injected with AAV8(Y733F)-hGRKl-mGCl and an age-matched, congenic GC1+/+ (control) mouse were sacrificed, their eyes enucleated and placed in IX PBS. Retinas were immediately dissected and processed as follows. Individual retinas were solubilized in PBS (137 mM NaCl, 2.7 mM KC1, 10 mM Na₂HPO₄, 1.8 mM KH2PO4) with 1% Triton X-100 and complete protease inhibitor (Roche) for 1 hour at 4°C, followed by centrifugation at 14000 rpm. The protein concentration of the supernatant

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2018203034 01 May 2018 was determined by BCA (Pierce) and 15 pg of each sample was separated on a 12% polyacrylamide gel (Bio-Rad) and transferred onto Immobilon-FL membranes for I hour in transfer buffer (25 mM Tris, 192 mM glycine) containing 15% methanol. Blots were treated with blocking buffer (Li-Cor) and labeled for 1 hour with a mouse monoclonal antibody recognizing GC1 (IS4, 1:3000, provided by Dr. Kris Palcweski, Case Western University, USA.) and rabbit polyclonal antibodies raised against GCAP1 (pAb UW14, 1:25,000, provided by Dr, Wolfgang Baehr, University of Utah) and β-actin (1:5000, Abeam). Secondary antibodies (goat anti-mouse lg conjugated to CW800 and goat anti-rabbit conjugated with IR680) were applied for 1 hour and blots imaged with an Odyssey Infrared Imaging System (Licor, Lincoln, NE, USA).

mRNA quantification by rtPCR, Retinal Genome Recovery and Optic Nerve IHC [00215) Individual treated eyes with optic nerve attached were harvested from GCIKO mice 1 year post-treatment with either AAV8(Y733F)-hGRKl-mGCl or AAV5-smCBA-mGCl and an age-matched, untreated GC1 +/+ mouse. Retinas were dissected from the eye immediately and snap frozen in liquid nitrogen. Optic nerves were dissociated from the eyes, fixed in 4% paraformaldehyde overnight at 4 °C, immersed in 30% sucrose for 2 hours at 4°C, and then quick frozen in cryostat compound (Tissue Tek® OCT 4583; Sakura Finetek USA, Inc., Torrance, CA, USA) in a bath of dry ice/ethanol. Optic nerves were sectioned at 10 pm and stained according to previously described methods (Boye et al., 2010). Retinas were homogenized in 350 mL of Buffer RLT (RNeasy® Protect Mini Kit, Qiagen, Inc., Valencia, CA, USA) plus BME for 45 sec. Samples were centrifuged and the lysate was split in half (one half designated for genome recovery and the other half for RNA extraction) (Traint and Whitehead, 2009). Genome recovery was performed as described above. RNA extraction was performed with an RNeasy® Protect Mini Kit (Qiagen, Inc.). RNA was reverse transcribed (iScript® cDNA synthesis kit. Biorad Laboratories, Hercules, CA, USA) and used

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2018203034 01 May 2018 in real-time PCR (iQ SYBR® Green Supermix and MyiQ real-time PCR detection system interfaced with iCycler® thermal cycler, Biorad Laboratories) to measure the following retinal specific mRNAs: guanylate cyclase-1 (GC1), guanylate cyclase activating protein-1 (GCAP 1), cone transducin a (GNAT2), rod cGMP-specific 3’,5’ cyclic phosphodiesterase subunit alpha (PDE6a) and the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), (00216) Primer pairs for GCAP1, GNAT2, PDEaand GAPDH were identical to those used by Baehr et al. (2007). Primers for murine GC1 (forward primer: 5'-GACCCTTCCTGCTGGTTCGATCCA-3' [SEQ ID NO:16], reverse primer: 5-CTGCATGTGTAGCAGCCTGTGCCTC-3' [SEQ ID NO:17]) were designed to flank exon 5, the site of gene disruption in the GCIKOmouse (Yang et al., 1999) and generate an amplicon of 151 bp. PCR produced appropriately sized amplicons in GC1 +/+ and AAVmGCl-treated GCIKO retina samples, but not in untreated GCIKO retina as expected. Amplicon identity was verified by restriction digest with Srwl (NEB) which cleaves within the target sequence to yield fragments of 56 bps and 95 bps. rtPCR with GC1 and GAPDH primers on dilution series of reverse transcribed DNA (from both GC1 +/+ and AAV-mGCltreated GCIKOretina samples) resulted in similar slopes, indicating suitability of GC1 primers for quantifying both endogenous and vector mediated GC1 message (FIG, 20A and FIG. 20B).

[00217( Results are the average of 3 replicate reactions and were calculated using the 2'^ωστ method (Livak and Schmittgen, 2001) with GAPDH signal used to normalize samples and the GCI +/+ sample serving as the calibrator. Standard deviations were calculated from the 3 replicate reactions done for each sample. Data is presented as the fold change in mRNA levels relative to the GCI +/+ sample.

Results

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Long-Term, Photoreceptor-Specific GC1 Expression:

[00218] Immunostaining with an antibody directed against GC1 revealed that AAV-vectored therapeutic protein expression persisted exclusively in photoreceptors of treated GC1KO mice for a significant fraction of the animal’s lifetime; AAV8(Y733F)-hGRKl-mGCl for at least 7 months, AAV5-smCBA-mGCl for at least 10 months, and AAV5-hGRKl-mGCl for at least 10 months (FIG. 21A and FIG. 2IB), GC1 expression was limited to the outer segments of rods and cones treated with AAV8(Y733F)-hGRKl-mGCl vector whereas it was found in both outer segments and more rarely in photoreceptor cell bodies of eyes treated with AAV5smCBA-mGCl, a result consistent with the strength of this ubiquitous promoter relative to photoreceptor-specific, hGRKl (Beltran et at, 2010). Two examples of retinal thinning were observed. The first was a GC1 KOretina treated with AAV5-smCBA-mGCl (4.69 χ 10⁹ total vector genomes delivered). The outer nuclear layer (ONL) was slightly thinned relative to that seen in naive GC1 KO or GC1+/+ control retinas (both 8 months of age). This may be a result of over-expression of GO mediated by the smCBA promoter (Beltran et at, 2010). [002191 The second involved a GC1 KO retina treated with the more concentrated AAV5hGRKl-mGCl and as before showed photoreceptor-specific GC1 expression but with profound thinning of the outer nuclear layer. It should be noted that this vector was the most concentrated of the three evaluated in this study (4.12 χ 1O^1U vector genomes delivered versus 4.69 χ 10⁹ and 1.08 χ 10¹⁰, for the AAV5-smCBA-mGCl prep and AAV8(Y733F)-hGRKlmGCl, respectively), and again highlights that over expression of GO may be the cause of the observed thinning. At a minimum, these results suggest that a dose limiting toxicity may be observable in the mouse. GC1 expression was absent from the untreated GO KO retina (FIG.21AandFIG.21B).

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Long Term Cone Photoreceptor Survival is Achieved by AA V- Vectored GC1 [002201 Cone photoreceptors in treated and untreated GC1KO mice as well as GC1+/+ controls were identified by staining for mouse cone arrestin. Retinal cross sections from mice sacrificed for the final biodistribution study and retinal whole mounts from a GC1KO mouse ! 1 months post-treatment with AAV5-smCBA-mGCl (right eye only) were analyzed. Here it was shown that cone photoreceptor densities were markedly reduced in untreated GC1KO retinas by 10 months of age and confirm previous reports that cones are lost in a topographically specific manner in this mouse model (Coleman et al., 2004) (FIG. 21A and

FIG. 2IB). Whole mount analysis revealed the 11 month old, untreated retina exhibited a sparse cone density, with residual cones found exclusively in superior retinal regions whereas the partner, P14-treated retina retained much higher cone density throughout, with the exception of a small patch of temporal retina which likely was not exposed to vector during the subretinal injection and therefore did not contain transgene product. Compared to that seen in AAV5-treated retinas, cone densities and structure in retinal cross sections of

AAV8(Y733F)-treated mice appeared qualitatively most similar to that seen in the normal,

GC1+/+ retina (FIG. 21A and FIG. 2IB). While their densities were increased relative to untreated controls, cones in AAV5-treated retinas appeared slightly disorganized, a result likely due to the slight overall disorder/thinning of the outer nuclear layers in these mice.

Long-term Restoration of Photoreceptor Function (ERG) in AAV-Treated GC1KO Mice [00221] In the previous examples, cone-mediated function could be restored to GC1KO mice for 3 months following Pl4 delivery of AAV5-smCBA-mGC 1 or AAV5-hGRKl-mGCl (Boye et at, 2010). Average photopic b-wave amplitudes in treated mice were partially restored at 4 weeks post-injection and remained stable throughout that study. In the present example, cone-mediated responses out to 9 months post-treatment were compared in GC1 KO mice injected between P14 and P25 with identical vectors used in the previous study. All

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2018203034 01 May 2018 remaining mice treated with AAV5-mGCI vector continued to exhibit measurable conemediated function out to at least 1 year post-treatment. Representative traces elicited at 12cds/m² from an individual mouse treated with AAV5-hGRKl-mGCl are shown in FIG. 22A and FIG. 22B. Cone responses were stable over time and were significantly higher than responses generated from untreated, contralateral controls (p< 0.001), suggesting that restoration of cone function is possible over the lifetime of the animal (FIG. 22A). Consistent with the previous example, the level of restoration achieved following delivery of the photoreceptor-specific promoter (hGRKl)-containing vector was not significantly different from that achieved with the ubiquitous promoter (smCBA)-containing vector at any posttreatment time point. Representative traces reveal that the kinetics of the restored cone ERG appeared normal throughout the course of the study (FIG. 22B). In addition, it was shown in this example that cone photoreceptor function was stably restored for at least 6 months following injection with AAV8(Y733F)-hGRKl-mGCl.

(00222) Cone b-wave amplitudes in GC1KO mice injected with this strong, fast-acting AAV8 tyrosine capsid mutant were higher than those seen in GC1KO mice injected with either AAV5 vector at every time point evaluated. At 6 months post-treatment, the latest time point in which all vectors could be compared in parallel, there was a significant difference between cone b-wave amplitudes in AAV8(Y733)-hGRKl-mGO vs. AAV5-hGRKI-mGCl -treated mice (p ~ 0.033) and AAV(Y733F)-hGRKl-mGCl vs, AAV5-smCBA-mGCl-treated mice (p = 0.025). A representative trace recorded 9 months post-injection with AAV8(Y733F)hGRKl-mGCl (n = 1) was noticeably smaller than that recorded at 6-months’ post-injection. [00223( Due to the inter-mouse variability in untreated GC1 KO rod responses (50-70% of WT by 5 months of age (23), statistical comparison of average rod responses of treated vs. untreated eyes is problematic. However, within an animal, rod ERG amplitudes are nearly equal between partner eyes, therefore we calculated the average intra-mouse rod a- and bwave amplitude ratios for treated versus untreated eyes and then plotted these ratios over time

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2018203034 01 May 2018 (FIG. 23A and FIG, 23B). AAV-mediated restoration of rod function is indicated by ratios with a value > t .0. FIG. 23A and FIG, 23B show that, with the exception of one time point (4 months post-treatment), the average ratios of rod b-wave amplitudes in AAV8(Y733F)hGRK.l-mGCl-treated uv. untreated eyes were all >1,0. Ratios of AAV5-treated v.v. untreated eyes were only occasionally >1.0. Similarly, rod a-wave ratios were consistently higher in AAV8(Y733F)-hGRK I-mGCl-treated mice, whereas they often declined following treatment with either AAV5 vector (FIG. 23 B). These results suggest that while the therapeutic effects on rods were subtle, AAV8(Y733F) conferred the most robust rodmediated functional improvement to the GC1KO mouse (FIG. 23B). Representative rodmediated scotopic ERG traces elicited by a 1 cds/m² stimulus were demonstrated in an AAV8(Y733F)-hGRKl-mGCl-treated GC1KO mouse (6 months post-treatment), the untreated contralateral control eye and an age-matched GC1+/+ control. AAV8(Y733F)mediated improvements in rod ERG amplitudes are clear in this example and indicate that aside from the sub-wild type amplitudes, treated eye response kinetics resemble that seen in the GC1 +/+ control.

Vector Biodistribution:

[00224] Biodistribution studies were performed in GC1KO mice treated with each vector to establish whether AAV5 or AAV8(Y733F)-delivered vector genomes could be detected in the optic nerves and/or brains of treated mice after a period of months. Mice injected with AAV5 vectors were evaluated at 7 (n = 2) and 10 (n = 5) months post-treatment and mice injected with AAV8(Y733F)-hGRKl-mGCl were evaluated at 4 (n = 1) and 7 (n = 1) months posttreatment. The optic nerves from injected and uninjected eyes were examined as well as portions of left and right brain that contained visual pathways. AAV5 vectors were injected in the right eyes of GC 1 KO mice. Accordingly, vector genomes were detected in the right optic nerve of AAV5-treated mice at both 7 and 10 months post-injection. At 7 months post94

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2018203034 01 May 2018 injection, vector genomes were also detected in the left brain of one mouse injected with AAV5-hGRKl-mGCl. No vector genomes were detected from the right brain of that animal. The observation that right (injected) optic nerve and left brain were positive is anatomically consistent since the left hemisphere is predominantly “wired” to the right eye.

[00225] By 10 months post-injection, AAV5 delivered vector genomes were still detected in right (injected) optic nerve but were absent from both brain hemispheres, AAV8(Y733) vector was injected into the left eyes of GC1KO mice. Accordingly, AAV8(Y733F)delivered vector genomes were detected in the left optic nerves at both 4 and 7 months postinjection. At no time point were vector genomes in the AAV8(733)-treated mouse detected in either brain hemisphere. A higher average number of vector genomes were detected in optic nerves of eyes injected with AAV5-GRKl-mGCI compared to AAV5-smCBA-mGCl. This result is likely due to the higher titer of the former (4.12 χ 10¹³ vg/mL) compared to the latter (4.69 x 10¹² vg/mL).

[00226] In addition, only AAV5-hGRKl-mGCl-delivered genomes were detected in brain tissue over the course of this study, another observation likely due to the relatively high titer of this vector. Despite the fact that the titer of AAV8(Y733F)-hGRKl-mGCl vector used (1.08 χ 10¹³ vg/mL) was less than that of the AAV5-hGRKl-mGCl vector, a higher average number of vector genomes was detected in optic nerves of AAV8(Y733F)-treated eyes. While AAV5 is known to be ineffective for transducing ganglion cells of the mouse retina (Stieger et al, 2008), it was shown that AAV8 does transduce this cell type (Jacobson et al., 2006). Some exposure of vector to retinal ganglion cells is expected as the syringe transverses the inner retina during subretinal injection and because the ratio of injection volume to total eye size is high in mouse. The higher number of vector genomes detected in optic nerves of AAV8(Y733F)-treated eyes therefore could be due to the increased affinity of AAV8(Y733F), relative to AAV5, for retinal ganglion cells. As expected, no AAV vector genomes were recovered from any tissue of naive GCI KO control mice.

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AAV-mGCl Treatment Restores Wild-type Levels of GC1 and GCA Pi to Treated GCIKO Retina [00227] At 7 months post-injection with AAV8(Y733F)-hGRKl-mGCl, treated and untreated retinas from one GC1KO mouse as well as one age-matched GC1+/+ control mouse were used to assay levels of GC1 and GCAPt protein expression. The goal of this experiment was not to compare GC1 levels across treatment groups but rather to compare levels of vectormediated GC1 expression to levels of GC1 in a wild type animal. Similarly we evaluated the effects of AAV-delivered GC1 on GCAP1 expression. As expected, GC1 protein was absent from the untreated eye of the GC1 KO mouse. In contrast, levels of GC1 in the AAV8(Y733F)-treated eye approached that seen in the normal, GC1+/+ control (Figure 4). Consistent with previous reports that GCAP1 is post-translationally downregulated in the GC1KO mouse, we show that GCAP1 was downregulated in untreated GC1KO retina relative to the GC1+/+ control (39). However, AAV8(Y733F)-mediated delivery of GC1 leads to an upregulation in GCAP1 expression in the treated GCIKO mouse retina. Levels of GCAP1 expression were also comparable to that seen in GC1+/+ controls.

[00228] In treated GCIKO mice, GC1 mRNA is present and GNAT2 mRNA levels are increased relative to untreated GCIKO mice. Using a GC1 primer pair that flanks the neomycin gene disruption located within Exon 5 of the GCIKOmouse (Timmers et al., 2001) it was possible to measure GC1 mRNA in both GC1 +/+ and vector-treated GCIKOmice. Interestingly a second GC1 primer pair targeted to exon 18 and 19 of GC1, well downstream of the gene disruption, produced a PCR product in the untreated GCIKO mouse sample and therefore these primers were not used. At one-year post-treatment, levels of GC1 mRNA in treated retinas were approximately seven-fold (AAV5-treated) and 14-fold [AAV8(YY733F)treated] higher than that seen in the age-matched GC1+/+ control mouse (FIG. 24A and FIG. 24B). By using a nucleic acid recovery technique that enabled homogeneous partitioning of the sample into 2 equal halves, one for RNA extraction and the other for DNA

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2018203034 01 May 2018 (Pang et al., 2011), albeit was possible to measure mRNA levels and determine the number of vector genomes within the same sample. It was found that high levels of GC1 mRNA in treated retinas corresponded to recovery of many vector genomes; 1.57 χ 10⁷ vector genomes/pg of DNA for AAV8(Y733F) and 4.7 χ 10⁶ vector genomes/pg for AAV5. Despite the high levels of GC1 mRNA in treated retinas, no GC1 expression was detected in optic nerves of treated eyes. This result further supports the notion that vectors evaluated in this study did not result in off-target transgene expression. Consistent with previous reports that the reduction of GCAP1 in GCIKO mice is post-translational (i.e., mRNA levels are unchanged), we found no substantial changes in the levels of GCAP1 mRNA across samples (FIG. 24A and FIG. 24B).

100229) As an initial estimate of treatment on other cone specific RNAs, several other transcripts were also evaluated in these samples. To establish a baseline for levels of cone transducin a (GNAT2), GNAT2 RNA was evaluated in untreated GCIKO samples and found to be reduced relative to GC1+/+ controls, a result likely due to the loss of cone photoreceptors in these retinas (FIG. 24A and FIG. 24B). In contrast, there were appreciable increases GNAT2 mRNA levels in eyes treated with either AAV5 or AAV8(Y733F) vectors, a result which further supports the notion that cone photoreceptors are preserved in AAVmGCl-treated GCIKO mice. Levels of rod PDE6a were relatively unchanged across samples likely because rod photoreceptors do not degenerate in the GCIKO mouse (FIG. 24A and FIG. 24B).

[00230) In conculsion, these studies demonstrate that persistent AAV-mediated GC1 expression is capable of restoring long term retinal function and preserving cone photoreceptors in the GCIKO mouse. Cohorts of AAV5- and AAV8(Y733F)-treated GCIKO mice were evaluated for ERG recovery for 9 months and 6 months post-injection, respectively, While the statistical comparison of cone ERG amplitudes did not continue beyond these time points due to dwindling sample sizes, all treated mice continued to exhibit

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2018203034 01 May 2018 functional (ERG) rescue, A variety of assays performed on subsets of these remaining mice all show clear indications of continuing therapy. This therapeutic longevity was validated on a number of different levels: 1) the existence of GC1 protein in treated eyes at 10 months post-treatment, 2) the restoration of cone function as measured by ERG at 12 months posttreatment, 3) the increased cone survival in treated eyes at 11 months post-treatment and 4) the recovery of vector genomes and GC1 mRNA in retinas at 12 months post-treatment. When viewed as individual, discrete analyses, the sample sizes used in these assays were often small. However when all are considered as correlates of therapeutic efficacy in mice exhibiting clear signs of functional rescue, the sample size is effectively much larger. Within this context, therefore, it appears that therapy persists beyond the period statistically evaluated for ERG rescue. This is the first demonstration of long-term therapy in an animal model of GC1 deficiency.

[00231] Restored cone ERGs were observed in AAV5 and AAV8(Y733)-treated GC1KO mice for at least 9 months and 6 months post-treatment, respectively. Responses were stable and significantly higher than untreated GC1KO cone responses throughout the course of the study. Recovery was most pronounced in mice treated with AAV8(Y733F) vector. Average cone b-wave amplitudes in AAV8(Y733F)-treated mice were consistently ~20pV higher than those recorded from GC1K.0 mice treated with standard AAV5 vectors (-55 μV vs. -35pV, respectively). At 6 months post-treatment, the latest time point that all vectors were statistically compared, this difference remained significant. This result confirms that an AAV8(Y733F) vector stably restored retinal structure and function to the rdlO mouse, a model refractory to treatment with standard AAV vectors.

[00232] Quantifying differences in rod amplitudes between treated and untreated eyes in the GC1KO mouse is complicated by the fact that rod function in this model is partially subserved by guanylate cyclase-2 (GC2) (Sun etal., 2010). Rod ERG responses are therefore variable from animal to animal (30-50% of normal). Therefore, unlike comparisons of treated

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2018203034 01 May 2018 and untreated cone responses, treated rod responses cannot be compared to a zero baseline. Nevertheless, paired GC1 KO eyes have comparable rod ERG amplitudes, and the intraanimal ratio of rod ERGs in partner eyes, one treated and the other untreated, provides a valid metric for evaluating treatment effects on rod function. Improvements in rod-mediated responses in AAV8(Y733F)-treated GC1 KO mice were observed more consistently than those recorded from AAV5-treated mice as indicated by comparing the intra-individual ratio of rod a- and b-wave amplitudes from the treated and untreated eye. This suggests that aggressive expression of GC1 in the GC1 KO eye can supplement the partial effect of GC2 on murine rod function, [00233| Long-term cone photoreceptor survival (11 months post-injection) was demonstrated by immunostaining treated and untreated retinal wholem ounts from one mouse treated with AAV5-smCBA-mGCl with an antibody directed against cone arrestin. Cones were identified throughout the treated GC1 KO retina. AAV5-smCBA-mGCl-treated retina also clearly contained more cones than the untreated eye which, consistent with previous reports, retained only a small fraction of cones in its superior hemisphere (Provost et at, 2005). While the preserved cones in treated GC1 KO retina were not examined on an ultrastructural level (e.g., electron microscopy), the observation that cones remained functional over time by ERG analysis suggests that their structure was intact. Long term preservation of cone photoreceptors mediated by therapeutic AAV-GC1 has obvious clinical relevance because it suggests the potential to preserve macular cones and restore usable daytime/color vision to patients with GC1 deficiency.

[00234J AAV-mediated GC1 expression persisted for at least 10 months post-treatment (the latest time point evaluated by 1HC), and was located exclusively in photoreceptors, regardless of the serotype used or whether a photoreceptor-specific (hGRKl) or ubiquitous (smCBA) promoter drove its expression. While transgene expression was limited to the target cell type, the hGRKl promoter was more specific in that it resulted in expression exclusively within the

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2018203034 01 May 2018 proper compartment of the target cell (photoreceptor outer segments). This result, along with other successful proof-of-concept studies utilizing this promoter suggests that the hGRKl promoter should be considered in the design of a clinical AAV vector targeting photoreceptors.

|00235] Immunostaining of transverse GC1 KO retinal sections at 10 months post-treatment with AAV5-smcBA-mGCl revealed moderate thinning of the ONL relative to the wild type and untreated GO KO controls. Additionally, in this retina GO was occasionally found in cell bodies of photoreceptors. It is possible that the strong, ubiquitous smCBA promoter drove expression of GC1 at levels that overwhelmed the trafficking machinery of some photoreceptors and that the accumulation of transgene product in photoreceptor cell bodies constituted a stress-initiated apoptosis in these cells. More dramatic ONL thinning was observed in one mouse injected with AAV5-hGRKl-mGCl. With an n of 1, it cannot be definitively conclude that retinal thinning was present in all mice treated with this vector. Nevertheless, consistent with the notion of overexpression toxicity, the titer of the AAV5hGRKl-mGCl vector was the highest of the three vectors evaluated in this study. However, it should also be noted that there was no accumulation of GC1 in photoreceptor cell bodies with the high titer AAV5-hGRKl-mGCl vector.

[00236] Despite the photoreceptor-exclusive nature of AAV-mediated GC1 expression, the inventors were interested in evaluating the spread of vector genomes to tissues outside the subretinal space. Importantly, these data were collected from ‘diseased’ animals. This is relevant based on evidence that the pattern of vector transduction is different in diseased vs. healthy retina (Kolstad et al., 2010). This would suggest that biodistribution patterns may also be different. For this reason, it was important to evaluate the spread of genomes within the rescued animal model itself (i.e., within subjects that exhibited clear ERG recovery). Although the sample size was limited, useful information was collected about the distribution of AAV5- and AAV8(Y733F)-delivered genomes in optic nerve and brain.

KM)

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2018203034 01 May 2018 [00237) This is the first evaluation of biodistribution for an AAV vector containing a capsid surface exposed tyrosine mutation. AAV5- and AAV8(Y733F)- delivered vector genomes were detected in the optic nerves of injected eyes at all time points assessed. At only one time point (7 months post-injection) were AAV5 vector genomes detected in the brain of a treated GC1KO mouse. Genomes were recovered only in the hemisphere opposite the injected eye. This result contrasts the finding by Provost et al., 2005 who reported a lack of AAV5delivered sequence in brains of subretinally-injected rats and dogs. By 10 months postinjection, no vector genomes were recovered from brains of AAV5- treated GC1 KO mice nor from brains of mice treated with AAV8(Y733F) at any time point. However, due to the relatively small number of mice analyzed, it cannot unequivocally be excluded that AAV5delivered genomes were present in brains at 10 months post-injection or that AAV8(Y733F) delivered genomes are never present in brains of treated GC1KO mice at any time.

[00238] Despite recovering vector genomes from optic nerves of treated eyes, immunostaining revealed a lack of GC1 expression in optic nerves of eyes treated with either AAV5-smCBAmGCl or AAV8(Y733F)-hGRKl -mGCl vectors. A previous study by Stieger,e/ al., (2005) detected transgene expression in optic nerves and brains of rats and dogs at 2 months and 4 weeks post-subretinal injection with AAV8 containing green fluorescent protein (GFP). Taking into account that the AAV8(Y733F) vector contained the photoreceptor-specific hGRKl promoter and the previous finding that GC1 expression was limited to photoreceptors even when under the control of a ubiquitous promoter like smCBA, a lack of GC1 expression in optic nerves is not unexpected. Stieger et al., (2005) incorporated the strong, ubiquitous CMV promoter into their vector to drive GFP, a protein which is capable of being stably expressed in a wide variety of tissues when delivered via viral vectors.

[00239) While both AAV5 and AAV8(Y733F) vectors were capable of providing long term therapy to the GC1KO mouse, there are apparent advantages associated with using AAV8(Y733F). First and foremost, AAV8{Y733F) with a photoreceptor-specific promoter

UH

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2018203034 01 May 2018 conferred significantly higher cone ERG responses to treated mice than either AAV5 vector. The reason for this may be due to the ability of AAV8 vectors to transduce areas outside of the injection bleb in rodent retina whereas the area of retina transduced by AAV5 remains largely confined to the bleb (47). Thus, AAV8(733F) may simply transduce on average a larger area of retina relative to AAV5 vectors and in tum result in more cone transduction and a robust full-field cone ERG response, through either or both an increased overall cone survival and/or an increased level of light response in each transduced cone.

Example 8 - Exemplary Mammalian GC1 Polypeptide Sequences [00240] Exemplary amino acid sequences useful in the practice of the present invention include, without limitation, one or more amino acid sequences that encode a biologicallyactive mammalian guanylate cyclase protein. Such sequences include, without limitation, those of human, non-human primate, murine, bovine, and canine origin, such as those guanylate cyclase proteins set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQIDNO:4, SEQ ID NO:5, SEQ1DNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10 and SEQ ID NO:11, hereinbelow:

[002411 Homo sapiens (human; GenPept Accession Number: NP 000171)

MTACARRAGGLPDPGLCGPAWWAPSLPRLPRALPRLPLLLLLLLL,QPPALSAVFTVGVLGPWACDP IFSRARPDLAARLAAARLNRDPGLAGGPRFEVALLPEpcrtpgslgavssalarvsglvgpvnpaa crpaellaeeagialvpwgcpwtqaegttapavtpaaealyallrafgwarvalvtapqdlwveag rslstalrarglpvasvtsmepldlsgarealrkvrdgprvtavimvmhsvllggeeqrylleaae elgltdgslvflpfdtihyalspgpealaalanssqlrrahdavltltrhcpsegsvldslrraqe RRELPSDLNLQQVSPLFGTIYDAVFLLARGVAEARAAAGGRWVSGAAVARHIREAQVPGFCGDLGG deeppfvlldtdaagdrlfatymldpargsflsagtrmhfprggsapgpdpscwfepnnicgggls PGLVFLGFLLWGMGLAGAFLAHYVRHRLLHMQMVSGPNKIILTVDDITFLHPHGGTSRKVAQGSR SS LGARSHSDIRSGP SQHLDS PNIGVYEGDRVWLKKF PGDQHIAIRPATKTAFSKLQELRHENVAL YLGLFLARGAEGPAALWEGNLAWSEHCTRGSLQDLLAQREIKLDWMFKSSLLLDLIKGIRYLHHR GVAHGRLKSRNCIVDGRFVLKITDHGHGRLLEAQKVLPEPPRAEDQLWTAPELLRDPALERRGTLA GDVFSLAIIMQEWCRSAPYAMLELTPEEWQRVRSPPPLCRPLVSMDQAPVECILLMKQCWAEQP ELRPSMDHTFDLFKNINKGRKTNIIDSMLRMLEQYSSNLEDLIRERTEELELEKQKTDRLLTQMLP psvaealktgtpvepeyfeqvtlyfsdivgfttisamsepiewdllndlytlfdaiigshdvykv etigdaymvasglpqrngqrhaaeianmsldilsavgtfrmrhmpevpvririglhsgpcvagwg LTMPRY CLFGDTVNTASRMESTGLPYRIHVNLSTVGILRALDSGYQVELRGRTELKGKGAEDTFWL VGRRGFNKPIPKPPDLQPGSSNHGISLQEIPPERRRKLEKARPGQFS (SEQ ID NO:1)

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2018203034 01 May 2018 [00242] Mus musculus (mouse; GenPept Accession Number: NP 032218)

MSAWLLPAGGLPGAGFCVPARQSPSSFSRVLRWPRPGLPGLLLLLLLPSPSALSAVFKVGVLGPWA CDPIFARARPDLAARLAANRLNRDFALDGGPRFEVALLPEPCLTPGSLGAVSSALSRVSGLVGPVN PAACRPAELLAQEAGVALVPWGCPGTRAAGTTAPAVTPAADALYVLLRAFRWARVALITAPQDLWV EAGRALSTALRAB-GLPVALVTSMETSDRSGAREALGRIRDGPRVRWIMVMHSVIjLGGEEQRYLLE AAEELALTEGSLVFLPFDTLHYALSPGPEALAAEVNSSQLRRAHEAVLTLTRRCPPGGSVQDSLRR AQEHQELPLDLNLKQVSPLFGTIYDAVFLLAGGVKRARTAVGGGWVSGASVARQVREAQVSGFCGV LGRTEEPSFVLLDTDASGEQLFATHLLDPVLGSLRSAGTPMHFPRGGPAPGPDPSCWFDPDVICNG GVEPGLVFVGFLLVIGMGLTGAFLAHYL.RHRLLHMQMASGPNKIILTLEDVTFLHPPGGSSRKWQ GSRSSLATRSASEIRSVPSQPQESTNVGLYEGDWVWLKKEPGEHHMAIRPATKTAFSKLRELRHEN VALYLGLFLAGTADSPATPGEGILAWSEHCARGSLHDLLAQREIKLEWMFKSSLLLDLIKGMRYL HHRGVAHGRLKSRNCVVDGRFVLKVTEHGHGRLLEAQRVLPEPPSAEEQLWTAPELLRDPSLERRG TLAGDVFSLAIIMQEWCRSTPYAMLELTPEEVIQRVRSPPPLCRPLVSMDQAPMECIQLMTQCMA EHPELRPSMDLTFDLFKSINKGRKTNIIDSNLRMLEQYSSNLEELIRERTEELEQEKQKTDRLLTQ MLPPSVAEALKMGTSVEPEYFEEVTLYFSDIVGFTTISAMSEPIEWDLLNDLYTLFDAIIGAHDV ykvetigeaymvasglpqrngqrhaaeianmsldilsavgsfrmrhmpevpvririglhsgpcvag WGLTMPRYCLFGDTVNTASRMESTGLPYRIHVNMSTVRILRALEQGFQMECRGRTELKGKGIEDT YWLVGRLGFNKFIPKPPDLQPGASNHGISLQEIPPERRKKLEKARPGQFTGK (SEQ ID NO:2) [00243J Rattus norvegicus (Norway rat; GenPept Accession Number: NP 077356)

MSAWLLPAGGFFGAGFCIPAWQSRSSLSRVLRWPGPGLPGLLLLLLLPSPSAFSAVFKVGVLGPWA CDPIFARARPDLAARLATDRLNRDLALDGGPWFEVTLLPEPCLTPGSLGAVSSALTRVSGLVGPVN PAACRPAELLAQEAGVALVPWGCPGTRAAGTTAPAVTPAADALYVLLKAFRWARVALITAPQELWV EAGRALSTALRARGLPVALVTSMVPSELSGAREALRRIRDGPRVRWIMVMHSVLLGGEEQRYLLE AAEELGLTDGSLVFLPFDTLHYALSPGPEALAAFVNSSKLRRAHDAVLTLTRRCPPGGSVQDSLRR AQEHQELPLDLDLKQVSPLFGTIYDAVFLLAGGVTRARAAVGGGWVSGASVARQMREAQVFGFCGI LGRTEEPSFVLLDTDAAGERLFTTHLLDPVLGSLRSAGTPVHFPRGAPAPGPDPSCWFDPDVICNG gvepglvfvgfllviwgltgaflahylrhrllhmqmvsgpnkiiltledvtflhpqggssrkvaq GSRSSLATRSTSDIRSVPSQPQESTNIGLYEGDWVWLKKFPGEHHMAIRPATKMAFSKLRELRHEN VALYLGLFLAGTADSPATPGEGILAWSEHCARGSLHELLAQRDIKLEWMFKSSLLLDLIKGMRYL HHRGVAHGRLKS RNCWDGRFVLKVTEHGHGRLLEAQRVLPE P PSAEEQLWTAPELLRDPALERRG TLAGDVFSLGIIMQEWCRSTPYAMLELTPEEVIQRVRSPPPLCRPLVSMDQAPMECIQLMAQCWA EHPELRPSMDLTFDLFKGINKGRKTNIIDSMLRMLEQYSSNLEDLIRERTEELEQEKQKTDRLLTQ MLPPSVAEALKMGTSVEPEYFEEVTLYFSDIVGFTTISAMSEPIEWDLLNDLYTLFDAIIGSHDV YKVETIGDAYMVASGLPQRNGQRHAAEIANMSLDILSAVGSFRMRHMPEVPVRIRIGLHSGPCVAG WGLTM PRYCLFGDT VNTASRMESTGLPYRI H VNMSTVRILRALDQGFQMECRGRTE LKGKGVEDT YWLVGRVGFNKPIPKPPDLQPGASNHGISLQEIPPERRKKLEKARPGQFTGK (SEQ ID NO:3) |00244] Bos taurus GCI (bovine; GenPept Accession Number: NP 776973)

MTACTFLAGGLRDPGLCAPTRWSPSPPGLPPIPFRPRLRLRPPLLLLLLLPRSVLSAVFTVGVLGP WACDPIFARARPDLAARLAASRLNHAAALEGGPRFEVALLPEPCRTPGSLGAVSSALTRVSGLVGP VNPAACRPAELLAQEAGVALVPWGCPGTRAAGTTAPWTPAADALYALLRAFRWAHVALVTAPQDL WVEAGHALSTALRARGLPVALVTSMEPSDLSGAREALRRVQDGPRVRAVIMVMHSVLLGGEEQRCL LEAAEELGLADGSLVFLPFETLHYALSPGPDALAVLANSSQLRKAHDAVLTLTRHCPLGGSVRDSL RRAQEHRELPLDLNLQQVSPLFGTIYDSVFLLAGGVARARVAAGGGWVSGAAVARHIRDARVPGFC GALGGAEEPSFVLLDTDATGDQLFATYVLDPTQGFFHSAGTPVHFPKGGRGPGPDPSCWFDPDTIC NGGVEPSWFIGFLLWGMGLAGAFLAHYCRHRLLHIQMVSGPNKIILTLDDITFLHPHGGNSRKV AQGSRTSLAARSISDVRSIHSQLPDYTNIGLYEGDWVWLKKFPGDRHIAIRPATKMAFSKIRELRH ENVALYLGLFLAGGAGGPAAPGEGVLAWS EHCARGS LQDLLAQRDIKLDWMFKSSLLLDLIKGIR YLHHRGVAHGRLKSRNCWDGRFVLKVTDHGHGRLLEAQRVLPEPPSAEDQLWTAPELLRDPVLER RGTLAGDVFSLGIIMQEWCRSAPYAMLELTPEEWKRVQSPPPLCRPSVSIDQAPMECIQLMKQC WAEQPELRPSMDRTFELFKSINKGRKMNIIDSMLRMLEQYSSNLEDLIRERTEELELEKQKTDRLL tqmlppsvaealkmgtpvepeyfeevtlyfsdivgfttisamsepiewdllnelytlfeaiigsh

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DVYKVETIGDAYMVASGLPQRNGHRHAAEIANMAEDILSAVGTFRMRHMFEVPVRIRIGLHSGPCV

AGWGLTMPRYCLFGDTVNTASRMESTGEPYRIHVNRSTVQILSAENEGELTEVRGRTELKGKGAE

ETYWLVGRRGFNKPIPKPPDLQPGASNHGISLHEIPPDRRQKLEKARPGQFSGK (SEQ ID NO:4) [00245] Canis lupus familiaris (canine; GenPept Accession Number: NP 001003207)

MSACALLAGGLPDPRLCAPARWARSPPGVPGAPPWPQPRLRLLLLLLLLPPSADSAVFTVGVLGPW ACDPIFARARPDLAARLAAARLNRDAALEDGPRFEVTLLPEPeRTPGSLGAVSSALGRVSGLVGPV N PAACRPAELLAQEAGVALVPWSCPGTRAGGTTAPAGTPAADALYALLRAFRWARVAEI tapqdlk VEAGRAESAALRARGLPVALVTTMEPSDLSGAREALRRVQDGPRVRAVIMVMHSVLLGGEEQRCEL QAAEELGLADGSLVFLPFDTLHYALSPGPEALAVLANSSQLRRAHDAVLILTRHCPPGGSVMDNLR RAQEHQELPSDLDLQQVSPFFGTIYDAVLLLAGGVARARAAAGGGWVSGATVAHHIPDAQVPGFCG TLGGAQEPPFVLLDTDAAGDREFATYMLDPTRGSLESAGTPVHFPRGGGTPGSDPSCWFEPGVICN GGVEPGLVFLGFLLWGMGLTGAFLAHYLRHRLLHIQMVSGPNKIILTLDDVTFLHPHGGSTRKW QGSRSSLAARSTSDIRSVPSQPLDNSNIGLFEGDWVWLKKFPGDQHIAIRPATKTAFSKLRELRHE NWLYLGLFLGSGGAGGSAAGEGVLAWSEHCARGSLHDLLAQRDIKLDKMFKSSLLLDLIKGMRY LHHRGVAHGRLKSRNCWDGRFVLKVTDHGHARLMEAQRVLLEPPSAEDQLWTAPELLRDPALERR GTLPGDVFSLGIIMQEWCRSAPYAMLELTPEEWERVRSPPPLCRPSVSMDQAPVECIQLMKQCW AEHPDLRPSLGHIFDQFKSINKGRKTNIIDSMLRMLEQYSSNLEDLIRERTEELELEKQKTDRLLT QMLPPSVAEALKMGTPVEPEYFEEVTLYFSDIVGFTTISAMSEPIEWDLLNDLYTLFDAIIGSHD VYKVETIGDAYMVASGLPQRNGQRHAAEIANMALDILSAVGSFRMRHMPEVPVRIRIGLHSGPCVA GWGLTM PRYCLFGDTVNTASRMESTGLPYRIHVNMSTVRILHALDEGFQTEVRGRTELKGKGAED TYWLVGRRGFNKPIPKPPDLQPGASNHGISLQEIPLDRRWKLEKARPGQFSGK (SEQ ID NO:5) [00246] Macaca mulatto (Rhesus macaque; predicted sequence from XP 001111670)

MTACARRAGGLPDPRLCGPARWAPALPRLPRALPRLPLLLLLLLLQPPALSAVFTVGVLGPWACDP IFSRARADEAARLAAARLNRDPDEAGGPRFEVALEPEPCRTPGSLGAVSSALTRVSGLVGPVNPAA CRPAELLAEEAGIALVPWGCPGTQAAGTTAPALTPAADALYALLRAFGWARVALVTAPQDLWVEAG HSLSTALRARGLPVASVTSMEPLDLSGAREALRKVRDGPRVTAVIMVMHSVLLGGEEQRYLLEAAE ELGLTDGSLVFLPFDTVHYALSPGPEALAALANSSQLRRAHDAVLTLTRHCPSEGSVLDSLRRAQE RRELPSDLNLQQVSPLFGTIYDAVFLLVRGVAEARAAAGGRKVSGAAVARHVWDAQVPGFCGDLGG DEEPPFVLLDTDAVGDRLFATYMLDPTRGSLLSAGTPMHFPRGGSAPGPDPSCWFDPNNICGGGLE PGLVFLGFLLWGMGLAGAFLAHYVRHQLLHIQMVSGPNKIILTVDDITFLHPHGGTSRKVAQGSR SS LAARSMSEVRS G PSQPTDS PNVGVYEGERVWLKKF PGDQHIAlRPATKTAFS KLQELRHENVAL YLGLFLAQGAEGPAALWEGNLAWSEHCTRGSLQDLLAQREIKLDWMFKSSLLLELIKGIRYLHHR GVAHGRLKSRNCIVDGRFVLKITDHGHGRLLEAQKVLPEPPRAEDQLWTAPELLRDPALERRGTLA GDVFSLAIIMQEWCRSAPYAMLELTPEEWQRVRSPPPLCRPLVSMDQAFVECIHLMKQCWAEQP ELRPSMDHTFDLEKNINKGRKTNIIDSMLRMLEQYSSNLEDLIRERTEELELEKQKTDRLLTQMLP PSVAEALKTGTPVEPEYFEQVTLYFSDIVGFTTISAMSEPIEWDLLNDLYTLFDAIIGSHDVYKV ETIGDAYMVASGLPQRNGQRKAAEIANMSLDILSAVGTFRMRHMPEVPVRIRIGLHSGPCVAGWG LTMPRYCLFGDTVNTASRMESTGLPYRIHVNLSTVGILRALDSGYQVELRGRTELKGKGAEDTFWL VGRRGFNKPIPKPPDLQPGSSNHGISLQEIPPERRRKLEKARPGQFS (SEQ ID NO:6).

]00247] Pongo abelti (Sumatran Orangutan; predicted sequence from XP_002827037)

MTACARRAGGLPDPGLCGPARWAPSLPRLPRALPRLPLLLLLLLLQPPALSAVFTVGVLG PWACDPIFSRARPDLAARLAAARLNRDPGLAGGPRFEVALLPEPCRTPGSLGAVSSALAR VSGLVGPVNPAACRPAELLADNPGIALVPWGCPWTQAEGTTAPCVTPAAEALYALLRAFG WARVALVTAPQDLWVEAGRSLSTALRARGLPVASVTSMEPLDLSGAREALRKVRDGPRVT AVIMVMHSVLLGGEEQRYLLEAAEELGLTDGSLVFLPFDTIHYALSPGPEALAALANSSQ LRRAHDAVLTLTRHCPSEGSVLDSLRRAQERRELPSDLNLQQVSPLFGTIYDAVFLLARG VAEAWAAAGGRWVSGAAVARHIRDAQVPGFCGDLGGDGEPPFVLLDTDAAGDRLFATYML DPARGSFLSAGTRMHFPRGGSAPGPDPSCWFDPNNICGGGLEPGLVFLGFLLWGMGLAG AFLAHYVRHRLLHIQMVSGPNKIILTVNDITFLHPHGGTSRKVAQGSRSSLAARSMSDIR

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SGPSQ PLDS PNVGVYEGDRVWLKKFPGDQHIAIRPATKTAFS KLQELRHENVALYLGLFL ARGAEGPAALWEGNLAWSEHCTRGSLQDLLSQREIKLDWMFKSSLLLDLIKGIRYLHHR GVAHGRLKSRNCrVDGRFVLKITDHGHGRLLEAQKVLPEPPRAEDQLWTAPELLREPALE RRGTLAGDVFSLAIIMQEWCRSAPYAMLELTPEEWQRVRSPPPLCRPLVSMDQAPVEC IHLMKQCWAEQPELRPSMDHTFDLFKNINKGRKTNIIDSMLRMLEQYSSNLEDLIRERTE ELELEKQKTDRLLTQMLPPSVAEALKTGTPVEPEYFEQVTLYFSDIVGFTTISAMSEPIE WDLLNBLYTLFEAIIGSHDVYKVETIGDAYMVASGLPQRNGQRHAAEIANMSLDILSAV GTFRMRHMPEVPVRIRIGLHSGPCVAGWGLTMFRYCLFGDTVNTASRMESTGLPYRIHV NLSTVGILRALESGYQVELRGRTELKGKGAEDTFWLVGRRGFNKPIPKPPDLQPGSSNHG ISLQEIPPERRRKLEKARPGQFS (SEQ ID NO:7) [002481 Callithrixjacchus (while tufted-ear marmoset; predicted sequence from XP_002747985)

MTACARRAGGLPDPGLCGPARWAPALSRLPRALPRLPLLLLLLLLQPPALSAQFTVGVLG PWACDPIFSRARPDLAARLAAARLNRDPSLAGGPRFEVALLPEPCRTPGSLGAVSSALAR VSGLVGPVNPAACRPAELLAEEAGIALVPWGCPGTQAAGTTAPVVTPAADALYALLRAFG WARVALVTAPQELWVEAGLSLSTALRARGLPWSVTSMEPLDLSGAREALRKVRNGPRVT AVIMVMHSVLLGGEEQRYLLEAAEELGLTDGSLVFLPFDTIHYALSPGREALAALVNSSQ LRRAHDAVLTLTRHCSSEGSVLDSLRKAQQRRELPSDLNLEQVSPLFGTIYDAWLLARG VADARAAVGGRWVSGAAVARHVWDAQASGFCGDLGRDEEPSFVLLDTDAAGDQLFATYML DPARGSLLSAGTPMHFPRGGPAPGPDPSCWFDPNNICDGGLEPGFIFLGFLLWGMGLAG ALLAHYVRHQLLHIQMVSGPNKIILTVDEITFLHPHGGASRKVAQGSRSSLAAHSTSDIR SGPSQ PSDSPNIGVYEGDRVWLKKFPGEQHIAIRPATKTAF S KLQELRHENVALYLGLFL AQGAEGPAALWEGNLAWSEHCTRGSLQDLLAQREIKLDWMFKSSLLLDLIKGIRYLHHR GVAHGRLKSRNCIVDGRFVLKITDHGHGRLLEAQKVLPEPPKAEDQLWTAPELLRDPALE RRGTLAGDVFSLGIIMQEWCRSAPYAMLELTPDEWQRVRSPPPLCRPFVSMDQAPVEC IHLMKQCWAEQPELRPSMDLTFDLFKNINKGRKTNIIDSMLRMLEQYSSNLEDLIRERTE ELELEKQKTDRLLTQMLPPSVAEALKTGTPVEPEYFEQVTLYFSDIVGFTTISAMSEPIE WDLLNDLYTLFDAIIGSHDVYKVETIGDAYMVASGLPQRNGQRHAAEIANMSLDILSAV GTFRMRHM PEVPVRIRIGLHSGP CVAGWGLTMPRYCLFGDTVNTAS RMESTGLPYRIHV NLSTVGILRALDSGYQVELRGRTELKGKGAEDTFWLVGRRGFNKPIPKPPDLQPGASNHG ISLQEIPPERRRKLEKARPGQFS(SEQ ID NO:8) [00249J Ailuropoda tnelanoleuca (giant panda; predicted sequence from XP_002921218)

MRACALLAGGLPYPRLCAPTRWAPARPGVSRALPWPRPRLRLLLLLLLRPPSVLSAVFTV GVLGPWACDPIFARARPDLXXXXXXXXXDALYVLLRAFRWARVALVTAPQDLWVEAGRAL SAALRARGLPVALVTTMEPSDLSGAREALRRVQHGPRVSAVIMVMHSVLLGGEEQRCLLQ AAEELGLADGSLVFLPFDTLHYALSPGPEALAALANSSQLRRAHDAVLTLTRHCPPGGSV MDSLRRAQERQELPSDLNLEQVSPLFGTIYDAVFLLAGGVARARAAAADSRVPGFCGALG GAEEPPFVLLDTDAAGDRFFATYVLDPTRGSLHSAGTPVHFPRGGGAPGPDPSCWFEPDS ICNGGVEPGLVFTGFLLWGMGLMGAELAHYVRHRLLHIQMVSGPNKIILTLDDITFLHP QGGSARKWQGSRSSLAARSTSDVRSVPSQPSDGGNIGLYEGDWVWLKKFPGSQHIAIRP ATKTAFSKLRELRHENVALYLGLFLGGGEGGSAAAGGGMLAWSEHCTRGSLHDLLAQRD IKLDWMFKSSLLLDLIKGMRYLHHRGVAHGRLKSRNCWEGRFVLKVTDHGKGRLLEAQK VLAEPPSAEDQLWTAPELLRDPALERRGTLAGDVFSLGIIMQEWCRSSPYAMLELSARE WQRVRSPPPLCRPSVSVDQAPAECIQLMKQCWAEQPBLRPSLDRTFDQFKSINKGRKTN IIDSMLRMLEQYSSNLEGLIRERTEELELEKRKTDRLRAASLPSSVAEALKMGTPVEPEY FEEVTLYFSDIVGFTTISAMSEPIEWELLNDLYTLFDAIIGSHDVYKVETIGEAYMVAS GLPQRNGQRHAAEIANMALDILSAVGSFRMRHMPEVPVRIRIGLHSGPCVAGWGLTMPR YCLFGDTVNTASRMESTGLPYRIHVNMSTVRILRALDEGFQTEVRGRTELKGKGAEDTYW LVGXXXXXXXXPIPKPPDLQPGASNHGISLQEIPLDRRQKLEKARPGQFSGK (SEQ IE NO:9} [00250] Monodelphis domestica (gray short-tailed opossum; predicted sequence from XP_001369029)

MLVPSINGLFHHPPWCFPPLPLPLFFLFLLLLLPVPVLPATFTIGVLGPWSCDPIFSRAR PDLAARLAATRMNHDQALEGGPWFEVILLPEPCRTS GS LGALS P S LARVSGLVG PVNPAA

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CHPAELLAQEAGVPLVPWGCPQGKARTTAPALPLALDALYALLRAFHWAKVALITAPQDL WVEAGQALAGGLRSRGLPVAMVTSLETTDLESAKNALKRVRDGPKVKVLIMVMHSVLLGG EEQRLLLEAAEELGLVEGTMVFLPFDTLHYALPPGPEALRFITNSSRLRKAHDAVLTLTR YCPKGSVSASLRQAQEHRELPLDLKPQQVSPLFGTIYDAIYLLAGAVAGAQVAGGGGWVS GAAVARHIPNTLVSGFCGDLGGTKEPPFVLLDTDGMRDQLLPTYTLDPAQGVLHHAGNPI HFPHGGQGPGPDPPCWFDPNVICSGGIEPRFILLVILIIIGGGLWATLAYYVKRQLLHA QMVSGPNKMILTLEEITFFPRQGSSSRKATEGSRSSLIAHSASDMRSIPSQPPDNSNIGM YEGDWVWLKKFPGEHYTEIRPATKMAFSKLRELRHENVAVQMGLFLAGSMEGAAAGGLGG GILAWSEYCSRGSLQDLLIQRDIKLDWMFKSS LLLDLIKGLRYLHHRGVAHGRLKSRNC WDGRFVLKITDHAHGRLLEAQRVSLEPPQAEDRLWTAPELLRNEALERQGTLQGDVFSV GIIMQEWCRCEPYAMLELTPEEIIQKVQSPPPMCRPSVSVDQAPMECIQLMKQCWAEQP DLRPNMDTTFDLFKNINKGRKTNIIDSMLRMLEQYS SNLEDLIRERTE ELELEKQKTDKL LTQMLPPSVAEALKLGIPVEPEYFEEVTLYFSDIVGFTTISAMSEPIEWDLLNDLYTLF DAIIGSHDVYKVETIGDAYMVASGLPKRNGQRHAAEIANMSLDILSSVGSFRMRHMPEVP VRIRIGLHSGPCVAGWGLTMPRYCLFGDTVNTASRMESTGLPYRIHVNLSTVKILQGLN egfqieirgrtelkgkgvedtywlvgrkgfdkpipippdllpgasnhgislqeipedrrk KLEKARPGQPLGK (SEQ ID NO: 10) (00251} Equus caballits (horse; predicted sequence from XP 001918412)

MVMHSVLLGGEEQRCLLEAAEELGLADGSLVFLPFDTLHYALSPGPEALAVLANNSQLRR AHDAVLTLTRHCPLGGSVLDSLRRAQEHQELPSDLNLQQVSFLFGTIYDAVYLLAGGVAR ARAAAGGSWVSGAAVAHHVRDAQVPGFCGALGGAEEPQFVLLDTDAAGDRLFATYMLDPT rgslwsagtpvhfprggrgpgpdpwcwfdpddicnggveprlvfigfllavgmglagvfl AHYVRHRLLHIQMASGPNKIILTLDDITFLHPQGGSSRKVIQGSRSSLAARSVSDIRSVP SQPMDSSNIGLYEGDWVWLKKFPGDQHIAIRPATKTAFSKLRELRHENVALYLGLFLAGG SSGAAAPREGMLAWSEHCARGSLHDLLAQRDIKLDWMFKS S LLLDLIKGMRYLHHRGVA HGRLKSRNCWDGRFVLKVTDHGHGRLLEAQKVLPEPPSAEDQLWTAPELLRDPALERQG TLAGDVFSLGIIIQEWCRSTPYAMLELTPEEWQRLQSPPPLCRPSVSMDQAPMECIQL MKQCWAEQPDLRPSMDRTFDLFKSINKGRKTNIIDSMLRMLEQYSSNLEDLIRERTEELE LEKQKTDRLLTQMLPPSVAEALKMGTPVEPEYFEEVTLYFSDIVGFTTISAMSEPIEWD LLNDLYTLFDAIIGSHDVYKVETIGDAYMVASGLPQRNGQRHAAEIANMALDILSAVGSF RMRHM PEVPVRIRIGLHSGPCVAGWGLTM PRYCLFGETVNTASRMESTGLPYRIHVNMS TVRILRALEEGFQVEVRGRTELKGKGVEDTYWLVGRRGFNKPIPKPPDLQPGASNHGISL QEIPPERRQKLEKARPGQFSGK (SEQ ID NO:11)

Example 9 - Sequence analysis of Known Mammalian GC1 Polypeptides [00252] All GC1 alignment data generated using amino acid sequence for the following species: Bos taunts (bovine; 1110 residues), Canis lupus familiaris (canine; 1109 residues), Mus muscuius (murine; 1108 residues), and Homo sapiens (human; 1103 residues). Positions of consensus and variable regions are based on numerical residues corresponding to Bos taurus as this is the longest GC1 protein, 1110 residues, and has no gaps in the alignment. [00253] Similarity graph of alignment of GC1 proteins from Bos taurus, Canis lupus familiaris, Mus muscuius, and Homo sapiens.

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2018203034 01 May 2018 [00254] GC1 consensus regions:

Amino acid positions: 44-49, 55-90, 98-155, 164-321, 464-549, 561-604, 620-761,

813-1026, 1045-1054, and 1060-1110.

[00255] Variable regions:

Amino acid positions: 4-43, 50-54, 91-97, 156-163, 322-463, 550-560, 605-619, 762812, 1027-1044, and 1055-1059.

[00256] Other notable regions of the GC1 consensus alignment include:

(1) Kinase homology domain: amino acid positions 531 to 541 of the consensus sequence (known to be essential for activity in photoreceptors- see, e.g., Bereta et al.,

2010).

(2) Phosphorylated serine residues within the kinase homology domain of murine GC1 protein (consensus/bovine position shown in parenthesis): 530 (532), 532 (534), 533 (535) and 538(540).

Example 10 - Nucleotide Sequence of the smCBA Promoter

1002571 The nucleic acid sequence of an illustrative human GRK1 (hGRKl) promoter which was used in the studies described above is shown below:

GGGCCCCAGAAGCCTGGTGGTTGTTTGTCCTTCTCAGGGGAAAAGTGAGGCGGCCCCTTGGAGGAAGG

GGCCGGGCAGAATGATCTAATCGGATTCCAAGCAGCTCAGGGGATTGTCTTTTTCTAGCACCTTCTTG

CCACTCCTAAGCGTCCTCCGTGACCCCGGCTGGGATTTAGCCTGGTGCTGTGTCAGCCCCGGTCTCCC

AGGGGCTTCCCAGTGGTCCCCAGGAACCCTCGACAGGGCCCGGTCTCTCTCGTCCAGCAAGGGCAGGG ACGGGCCACAGGCCAAGGGC (SEQ ID N0:12)

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2018203034 01 May 2018 [00258] The nucleic acid sequence of an illustrative smCBA promoter which was used in the studies described above is shown below:

AATTCGGTACCCTAGTTATTAATAGTAATCAATTACOGGGTCATTAGTTCATAGCCCATATATGGA

GTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT

GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGT ggactatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccc tattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactt tcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagccccacgtt ctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaatt attttgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcga ggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtt tccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagt cgctgcgacgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccgccccggctct gactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagc gcttggtttaatgacggcttgtttcttttctgtggctgcgtgaaagccttgaggggctccgggagc tagagcctctgctaaccatgttcatgccttcttctttttcctacagctcctgggcaacgtgctggt TATTGTGCTGTCTCATCATTTTGGCAAAG (SEQ ID KO:13)

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[00260] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those of ordinary skill in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Claims (40)

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

   1. A recombinant adeno-associated viral (rAAV) vector comprising at least a first polynucleotide that comprises a photoreceptor-specific human rhodopsin kinase promoter, or a ubiquitous truncated chimeric CMV-chicken β-actin promoter, operably linked to at least a first nucleic acid segment that encodes at least a first biologically active, retinal- specific mammalian guanylate cyclase polypeptide, wherein the rAAV vector is comprised within an adeno-associated viral 5 (AAV5) particle, wherein the at least a first biologically active, retinal- specific mammalian guanylate cyclase polypeptide comprises at least a first contiguous amino acid sequence region that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 1.
2. 2. The rAAV vector according to claim 1, wherein the at least a first biologically active, retinal-specific mammalian guanylate polypeptide comprises at least a first contiguous amino acid sequence region that is at least about 92% identical to the amino acid sequence of SEQ ID NO:1.
3. 3. The rAAV vector according claim 1 or claim 2, wherein the at least a first biologically active, retinal-specific mammalian guanylate cyclase polypeptide comprises at least a first contiguous amino acid sequence region that is at least about 95% identical to in the amino acid sequence of SEQ ID NO: 1.
4. 4. The rAAV vector according to any one of claims 1 to 3, wherein the at least a first biologically active, retinal-specific mammalian guanylate cyclase polypeptide comprises at least a first contiguous amino acid sequence region that is at least about 98% identical the amino acid sequence of SEQ ID NO: 1.
5. 5. The rAAV vector according to any one of claims 1 to 4, wherein the at least a first biologically active, retinal-specific mammalian guanylate cyclase polypeptide comprises the amino acid sequence of SEQ ID NO: 1.
6. 6. The rAAV vector according to any one of claims 1 to 5, wherein the vector is a self-complementary rAAV (scAAV).
7. 7. The rAAV vector according to any one of claims 1 to 6, wherein the photoreceptor-specific human rhodopsin kinase promoter comprises a nucleic acid

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   2018203034 01 May 2018 sequence that comprises an at least 30 contiguous base pair sequence from SEQ ID NO: 12, or the truncated chimeric CMV-chicken β-actin promoter comprises a nucleic acid sequence that comprises an at least 70 contiguous base pair sequence from SEQ ID NO: 13.
8. 8. The rAAV vector according to any one of claims 1 to 7, wherein the photoreceptor-specific human rhodopsin kinase promoter comprises a nucleic acid sequence that comprises an at least 40 contiguous base pair sequence from SEQ ID NO: 12, or the truncated chimeric CMV-chicken β-actin promoter comprises a nucleic acid sequence that comprises an at least 90 contiguous base pair sequence from SEQ ID NO: 13.
9. 9. The rAAV vector according to any one of claims 1 to 8, wherein the photoreceptor-specific human rhodopsin kinase promoter comprises a nucleic acid sequence that comprises an at least 60 contiguous base pair sequence from SEQ ID NO: 12, or the truncated chimeric CMV-chicken β-actin promoter comprises a nucleic acid sequence that comprises an at least 120 contiguous base pair sequence from SEQ ID NO: 13.
10. 10. The rAAV vector according to any one of claims 1 to 9, wherein the photoreceptor-specific human rhodopsin kinase promoter comprises the nucleic acid sequence of SEQ ID NO: 12, or the truncated chimeric CMV-chicken β-actin promoter comprises the nucleic acid sequence of SEQ ID NO: 13.
11. 11. The rAAV vector according to any one of claims 1 to 10, further comprising at least a first enhancer operably linked to the at least a first nucleic segment.
12. 12. The rAAV vector according to any one of claims 1 to 11, further comprising at least a first mammalian intron sequence operably linked to the at least a first nucleic segment.
13. 13. An isolated mammalian host cell comprising the rAAV vector in accordance with any one of claims 1 to 12.
14. 14. The isolated mammalian host cell according to claim 13, wherein the cell is a human host cell.
15. 15. A composition comprising:

    (1) (a) the rAAV vector in accordance with any one of claims 1 to 12;

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    2018203034 01 May 2018 (b) an isolated mammalian host cell comprising the vector; and (2) a pharmaceutically-acceptable buffer, carrier, vehicle, or diluent.
16. 16. The composition according to claim 15, further comprising a lipid, a liposome, a lipid complex, an ethosome, a niosome, a nanoparticle, a microparticle, a liposphere, a nanocapsule, or any combination thereof.
17. 17. The composition according to claim 15 or claim 16, formulated for administration to the human eye.
18. 18. The composition according to any one of claims 15 to 17, for use in therapy or prophylaxis.
19. 19. The composition according to any one of claims 15 to 18, for use in the therapy or prophylaxis of a human retinal dystrophy, disease, or disorder.
20. 20. The composition according to any one of claims 15 to 19, for use in the therapy or prophylaxis of Leber congenital amaurosis-1 (LCA1).
21. 21. A kit comprising:

    (1) a component selected from the group consisting of:

    (a) the rAAV vector in accordance with any one of claims 1 to 12;

    (b) the isolated mammalian host cell in accordance with claim 13 or claim 14;

    or (c) the composition in accordance with any one of claims 15 to 20; and (2) instructions for using the component in the diagnosis, prevention, treatment, or amelioration of one or more symptoms of a retinal dystrophy, disease, disorder, or abnormal condition in a human.
22. 22. The kit according to claim 21, wherein the retinal dystrophy is Leber congenital amaurosis-1 (LCA-1).
23. 23. Use of a composition in accordance with claim 15, in the manufacture of a medicament for diagnosing, preventing, treating or ameliorating a disease, disorder, dysfunction, or abnormal condition of a mammalian eye, or one or more symptoms thereof.

    120

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24. 24. Use according to claim 23, in the manufacture of a medicament for treating or ameliorating one or more symptoms of Leber congenital amaurosis-1 (LCA-1) in a human.
25. 25. A method for preventing, treating or ameliorating a disease, dysfunction, disorder, deficiency, or abnormal condition in a mammal, or one or more symptoms thereof, the method comprising administering to the mammal a composition comprising the rAAV vector in accordance with any one of claims 1 to 12, in an amount and for a time sufficient to prevent, treat or ameliorate the disease, dysfunction, disorder, deficiency, or abnormal condition in the mammal, or one or more symptoms thereof.
26. 26. The method according to claim 25, wherein the mammal has, is suspected of having, is at risk for developing, or has been diagnosed with at least a first retinal disorder, disease, or dystrophy.
27. 27. The method according to claim 25 or claim 26, wherein the mammal is at risk for developing, or has been diagnosed with a deficiency in biologically-active retinal guanylate cyclase protein, peptide, or polypeptide.
28. 28. The method according to any one of claims 25 to 27, wherein the mammal is a human neonate, newborn, infant, or juvenile that is at risk for developing or has been diagnosed with a congenital retinal dystrophy such as Leber congenital amaurosis-1 (LCA-1).
29. 29. A method for providing a mammal with a therapeutically-effective amount of a biologically-active mammalian guanylate cyclase peptide, polypeptide, or protein to a mammal in need thereof, comprising introducing into suitable cells of a mammal in need thereof, an effective amount of the rAAV vector in accordance with any one of claims 1 to 12, for a time sufficient to produce a biologically-active guanylate cyclase polypeptide therefrom in at least a first population of cells or at least a first tissue of the mammal into which the rAAV vector has been introduced.
30. 30. The method according to claim 29, wherein the mammal in need thereof has a defect, deficiency, or substantial absence of biologically-active retGC 1 protein in at least a first tissue of the mammal, when compared to the level of biologically-active retGC 1 protein in a normal mammal.

    121

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31. 31. The method according to claim 29 or claim 30, wherein a plurality of cells from the mammal are provided with the rAAV vector ex vivo or in vitro, and the method further comprises introducing the plurality of provided cells into at least a first tissue site within the body of the mammal.
32. 32. The method according to claim 31, wherein the plurality of provided cells is introduced into at least a first site within one or both eyes of the mammal.
33. 33. The method according to claim 31, wherein the plurality of provided cells is introduced into at least a first site within one or both eyes of the mammal by direct injection into the retina or the surrounding tissue.
34. 34. The method according to any one of claims 29 to 33, wherein introduction of the rAAV vector preserves cone photoreceptors, and restores cone-mediated function and visual behavior in the eye of the mammal.
35. 35. The method according to any one of claims 29 to 34, wherein a single introduction of the rAAV vector into the eye of the mammal preserves cone photoreceptors, and restores cone-mediated function and visual behavior in the mammal for a period of at least three months.
36. 36. The method according to any one of claims 29 to 35, wherein a single introduction of the rAAV vector into the eye of the mammal preserves cone photoreceptors, and restores cone-mediated function and visual behavior in the mammal for a period of at least six months.
37. 37. The method according to any one of claims 29 to 36, wherein a single introduction of the rAAV vector into the eye of the mammal preserves cone photoreceptors, and restores cone-mediated function and visual behavior in the mammal for a period of at least ten months.
38. 38. A method for increasing the level of biologically-active retGCl protein in one or more retinal cells of a mammal that has, is suspected of having, is diagnosed with, or is at risk for developing, LCA1, comprising introducing the rAAV vector in accordance with any one of claims 1 to 12, into at least a first population of retinal cells of the mammal, in an amount and for a time effective to increase the level of biologically-active retGC 1 protein in one or more retinal cells of the mammal.

    122

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39. 39. A method for treating or ameliorating one or more symptoms of retinal dystrophy in a mammal, the method comprising directly administering to the retina or sub-retinal space of the mammal an rAAV vector in accordance with any one of claims 1 to 12, in an amount and for a time sufficient to treat or ameliorate the one or more symptoms of retinal dystrophy in the mammal.
40. 40. The method according to claim 39, wherein the mammal has been diagnosed with Leber congenital amaurosis-1 (LCA-1).

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    2018203034 01 May 2018

    SEQUENCE LISTING <110> University of Florida Research Foundation, inc.

    Boye, Shannon E.

    Hauswirth, william w.

    Boye, Sanford L.

    <120> rAAV-Guanylate Cyclase Compositions and Methods for Treating Leber's Congenital Amaurosis-1 (LCAl) <130> 36689.309 <150> US 61/327,521 <151> 2010-04-23 <160> 18 <170> Patentln version 3.5 <210> 1 <211> 1103 <212> PRT <213> Homo sapiens <400> 1

    Met 1 Thr Ala Cys Ala 5 Arg Arg Ala Gly Gly 10 Leu Pro Asp Pro Gly 15 Leu cys Gly Pro Ala Trp Trp Al a Pro Ser Leu Pro Arg Leu Pro Arg Ala 20 25 30 Leu Pro Arg Leu Pro Leu Leu Leu Leu Leu Leu Leu Leu Gin Pro Pro 35 40 45 Ala Leu Ser Ala val Phe Thr Val Gly Val Leu Gly Pro Trp Ala Cys 50 55 60 Asp Pro ile Phe Ser Arg Ala Arg Pro Asp Leu Ala Ala Arg Leu Ala 65 70 75 80 Ala Ala Arg Leu Asn Arg Asp Pro Gly Leu Al a Gly Gly Pro Arg Phe 85 90 95 Glu val Ala Leu Leu Pro Glu Pro cys Arg Thr Pro Gly Ser Leu Gly 100 105 110 Al a val ser Ser Ala Leu Ala Arg val Ser Gly Leu val Gly pro val 115 120 125 Asn pro Ala Ala cys Arg pro Al a Glu Leu Leu Ala Glu Glu Ala Gly 130 135 140 lie Ala Leu Val Pro Trp Gly cys Pro Trp Thr Gin Ala Glu Gly Thr 145 150 155 160 Thr Ala Pro Ala Val Thr Pro Ala Ala Asp Ala Leu Tyr Ala Leu Leu

    165 170 175

    2018203034 01 May 2018

    Arg Ala Phe Gly Trp Ala Arg Val Ala Leu Val Thr Ala Pro Gln Asp 180 185 190 Leu Trp val Glu Ala Gly Arg Ser Leu Ser Thr Ala Leu Arg Ala Arg 195 200 205 Gly Leu Pro val Ala Ser val Thr Ser Met Glu Pro Leu Asp Leu Ser 210 215 220 Gly Ala Arg Glu Ala Leu Arg Lys val Arg Asp Gly Pro Arg Val Thr 225 230 235 240 Ala val lie Met val Met Hi s Ser val Leu Leu Gly Gly Glu Glu Gln 245 250 255 Arg Tyr Leu Leu Glu Ala Ala Glu Glu Leu Gly Leu Thr Asp Gly Ser 260 265 270 Leu val Phe Leu Pro Phe Asp Thr Ile Hi s Tyr Ala Leu Ser Pro Gly 275 280 285 Pro Glu Ala Leu Ala Ala Leu Ala Asn Ser Ser Gln Leu Arg Arg Ala 290 295 300 Hi s Asp Ala val Leu Thr Leu Thr Arg His cys Pro ser Glu Gly Ser 305 310 315 320 val Leu Asp Ser Leu Arg Arg Ala Gln Glu Arg Arg Glu Leu Pro Ser 325 330 335 Asp Leu Asn Leu Gln Gln Val Ser Pro Leu Phe Gly Thr lie Tyr Asp 340 345 350 Ala val Phe Leu Leu Al a Arg Gly val Ala Glu Ala Arg Ala Ala Al a 355 360 365 Gly Gly Arg Trp val Ser Gly Ala Ala val Al a Arg His lie Arg Asp 370 375 380 Ala Gln Val pro Gly Phe Cys Gly Asp Leu Gly Gly Asp Glu Glu Pro 385 390 395 400 Pro Phe val Leu Leu Asp Thr Asp Ala Ala Gly Asp Arg Leu Phe Ala 405 410 415 Thr Tyr Met Leu Asp Pro Ala Arg Gly Ser Phe Leu Ser Ala Gly Thr 420 425 430 Arg Met His Phe Pro Arg Gly Gly Ser Ala Pro Gly Pro Asp Pro Ser 435 440 445

    2018203034 01 May 2018

    Cys Trp Phe Asp Pro Asn Asn lie 450 455 Leu 465 Val Phe Leu Gly Phe 470 Leu Leu Ala Phe Leu Ala Hi s 485 Tyr val Arg val Ser Gly Pro 500 Asn Lys lie ile Leu Hi s Pro 515 Hi s Gly Gly Thr Ser 520 Ser ser 530 Leu Gly Ala Arg Ser 535 Met Gln 545 Hi s Leu Asp ser Pro 550 Asn lie Trp Leu Lys Lys Phe 565 Pro Gly Asp Thr Lys Thr Ala 580 Phe Ser Lys Leu Ala Leu Tyr 595 Leu Gly Leu Phe Leu 600 Ala Leu 610 Trp Glu Gly Asn Leu 615 Ala Gly 625 Ser Leu Gln Asp Leu 630 Leu Ala Met Phe Lys Ser Ser 645 Leu Leu Leu Leu Hi s Hi s Arg 660 Gly val Ala Hi s lie val Asp 675 Gly Arg Phe val Leu 680 Arg Leu 690 Leu Glu Ala Gln Lys 695 Val Asp Gln Leu Trp Thr Al a Pro Glu

    705 710

    Cys Gly Gly Gly 460 Leu Glu Pro Gly val val Gly 475 Met Gly Leu Ala Gly 480 Hi s Arg 490 Leu Leu Hi s Met Gln 495 Met Leu 505 Thr Val Asp Asp ile 510 Thr Phe Arg Lys val Ala Gln 525 Gly Ser Arg Ser Asp lie Arg 540 Ser Gly Pro Ser Gly val Tyr 555 Glu Gly Asp Arg Val 560 Gln Hi s 570 Ile Al a lie Arg Pro 575 Al a Gln 585 Glu Leu Arg His Glu 590 Asn val Ala Arg Gly Ala Glu 605 Gly Pro Al a val Val Ser Glu 620 His cys Thr Arg Gln Arg Glu 635 Ile Lys Leu Asp Trp 640 Asp Leu 650 Ile Lys Gly lie Arg 655 Tyr Gly 665 Arg Leu Lys Ser Arg 670 Asn cys Lys ile Thr Asp His 685 Gly Hi s Gly Leu Pro Glu Pro 700 Pro Arg Ala Glu Leu Leu Arg 715 Asp Pro Ala Leu Glu 720

    Arg Arg Gly Thr Leu Ala Gly Asp Val Phe Ser Leu Ala Ile Ile Met 3

    2018203034 01 May 2018

    725 730 735

    Gln Glu val val 740 Cys Arg Ser Ala Pro Tyr Ala Met Leu 745 Glu 750 Leu Thr Pro Glu Glu 755 Val Val Gln Arg val 760 Arg Ser Pro Pro Pro 765 Leu cys Arg Pro Leu Val Ser Met Asp Gln Ala Pro val Glu cys Ile Leu Leu Met 770 775 780 Lys Gln cys Trp Ala Glu Gln Pro Glu Leu Arg Pro Ser Met Asp Hi s 785 790 795 800 Thr Phe Asp Leu Phe Lys Asn lie Asn Lys Gly Arg Lys Thr Asn ile 805 810 815 Ile Asp ser Met Leu Arg Met Leu Glu Gln Tyr Ser Ser Asn Leu Glu 820 825 830 Asp Leu lie Arg Glu Arg Thr Glu Glu Leu Glu Leu Glu Lys Gln Lys 835 840 845 Thr Asp Arg Leu Leu Thr Gln Met Leu Pro Pro ser Val Ala Glu Ala 850 855 860 Leu Lys Thr Gly Thr Pro Val Glu Pro Glu Tyr Phe Glu Gln val Thr 865 870 875 880 Leu Tyr Phe Ser Asp lie val Gly Phe Thr Thr lie Ser Ala Met Ser 885 890 895 Glu Pro lie Glu Val val Asp Leu Leu Asn Asp Leu Tyr Thr Leu Phe 900 905 910 Asp Ala lie Ile Gly Ser His Asp Val Tyr Lys val Glu Thr lie Gly 915 920 925 Asp Ala Tyr Met val Al a ser Gly Leu Pro Gln Arg Asn Gly Gln Arg 930 935 940 Hi s Ala Ala Glu lie Ala Asn Met Ser Leu Asp lie Leu Ser Ala val 945 950 955 960 Gly Thr Phe Arg Met Arg His Met pro Glu val Pro val Arg lie Arg 965 970 975 lie Gly Leu His Ser Gly Pro cys val Ala Gly val val Gly Leu Thr 980 985 990

    Met Pro Arg Tyr Cys Leu Phe Gly Asp Thr val Asn Thr Ala Ser Arg 995 1000 1005

    2018203034 01 May 2018

    Met Glu 1010 Ser Thr Gly Leu Pro 1015 Thr Val 1025 Gly Ile Leu Arg Ala 1030 Leu Arg 1040 Gly Arg Thr Glu Leu 1045 Phe Trp 1055 Leu val Gly Arg Arg 1060 Pro Pro 1070 Asp Leu Gln Pro Gly 1075 Gln Glu 1085 lie Pro Pro Glu Arg 1090 Pro Gly 1100 Gln Phe ser

    Tyr Arg lie His Val Asn Leu Ser 1020

    Leu Asp Ser Gly Tyr Gln Val Glu 1035

    Lys Gly Lys Gly Ala Glu Asp Thr 1050

    Gly Phe Asn Lys Pro lie Pro Lys 1065

    Ser Ser Asn His Gly lie Ser Leu 1080

    Arg Arg Lys Leu Glu Lys Ala Arg 1095 <210> 2 <211> 1108 <212> PRT <213> Mus musculus <400> 2

    Met Ser Ala Trp Leu Leu Pro Ala Gly Gly 10 Leu Pro Gly Ala Gly 15 Phe 1 5 cys val Pro Ala Arg Gln Ser Pro Ser Ser Phe Ser Arg val Leu Arg 20 25 30 Trp Pro Arg Pro Gly Leu Pro Gly Leu Leu Leu Leu Leu Leu Leu Pro 35 40 45 Ser pro Ser Ala Leu Ser Ala Val Phe Lys val Gly Val Leu Gly Pro 50 55 60 Trp Ala Cys Asp Pro lie Phe Ala Arg Ala Arg Pro Asp Leu Ala Al a 65 70 75 80 Arg Leu Ala Ala Asn Arg Leu Asn Arg Asp Phe Al a Leu Asp Gly Gly 85 90 95 Pro Arg Phe Glu val Al a Leu Leu Pro Glu Pro cys Leu Thr Pro Gly 100 105 110 ser Leu Gly Ala Val Ser Ser Ala Leu Ser Arg val Ser Gly Leu val

    115 120 125

    2018203034 01 May 2018

    Gly Pro 130 Val Asn Pro Ala Ala 135 Cys Glu 145 Ala Gly val Ala Leu 150 Val Pro Ala Gly Thr Thr Ala 165 Pro Ala val val Leu Leu Arg 180 Ala Phe Arg Trp Pro Gin Asp 195 Leu Trp val Glu Al a 200 Arg Ala 210 Arg Gly Leu Pro Val 215 Ala Asp 225 Arg Ser Gly Ala Arg 230 Glu Ala Arg Val Arg Val val 245 Ile Met val Glu Glu Gin Arg 260 Tyr Leu Leu Glu Asp Gly Ser 275 Leu val Phe Leu Pro 280 Ser pro 290 Gly Pro Glu Al a Leu 295 Ala Arg 305 Arg Ala His Asp Al a 310 Val Leu Gly Gly Ser val Gin 325 Asp Ser Leu Leu Pro Leu Asp 340 Leu Asn Leu Lys lie Tyr Asp 355 Ala Val Phe Leu Leu 360 Thr Ala 370 Val Gly Gly Gly Trp 375 val val Arg Glu Ala Gin val ser Gly

    385 390

    Arg Pro Ala Glu 140 Leu Leu Ala Gin Trp Gly cys 155 Pro Gly Thr Arg Ala 160 Thr Pro 170 Ala Ala Asp Ala Leu 175 Tyr Ala 185 Arg val Ala Leu lie 190 Thr Ala Gly Arg Ala Leu ser 205 Thr Ala Leu Leu val Thr Ser 220 Met Glu Thr ser Leu Gly Arg 235 ile Arg Asp Gly Pro 240 Met Hi s 250 Ser val Leu Leu Gly 255 Gly Ala 265 Ala Glu Glu Leu Ala 270 Leu Thr Phe Asp Thr Leu His 285 Tyr Ala Leu Ala Phe Val Asn 300 Ser Ser Gin Leu Thr Leu Thr 315 Arg Arg Cys Pro Pro 320 Arg Arg 330 Al a Gin Glu His Gin 335 Glu Gin 345 val ser Pro Leu Phe 350 Gly Thr Ala Gly Gly Val Lys 365 Arg Ala Arg Ser Gly Al a Ser 380 Val Ala Arg Gin Phe cys Gly 395 val Leu Gly Arg Thr 400

    Glu Glu Pro Ser Phe Val Leu Leu Asp Thr Asp Ala Ser Gly Glu Gin 6

    2018203034 01 May 2018

    405 410 415

    Leu Phe Ala Thr His Leu Leu Asp Pro Val 425 Leu Gly Ser Leu 430 Arg Ser 420 Ala Gly Thr Pro Met Hi s Phe Pro Arg Gly Gly Pro Ala Pro Gly Pro 435 440 445 Asp Pro Ser cys Trp Phe Asp Pro Asp val Ile Cys Asn Gly Gly val 450 455 460 Glu Pro Gly Leu val Phe Val Gly Phe Leu Leu Val ile Gly Met Gly 465 470 475 480 Leu Thr Gly Ala Phe Leu Ala His Tyr Leu Arg His Arg Leu Leu Hi s 485 490 495 Met Gln Met Ala Ser Gly Pro Asn Lys ile Ile Leu Thr Leu Glu Asp 500 505 510 val Thr Phe Leu His Pro Pro Gly Gly Ser Ser Arg Lys val Val Gln 515 520 525 Gly Ser Arg ser Ser Leu Ala Thr Arg Ser Ala Ser Asp lie Arg ser 530 535 540 val Pro Ser Gln Pro Gln Glu Ser Thr Asn val Gly Leu Tyr Glu Gly 545 550 555 560 Asp Trp Val Trp Leu Lys Lys Phe pro Gly Glu Hi s His Met Ala Ile 565 570 575 Arg Pro Ala Thr Lys Thr Ala Phe Ser Lys Leu Arg Glu Leu Arg Hi s 580 585 590 Glu Asn val Ala Leu Tyr Leu Gly Leu Phe Leu Ala Gly Thr Ala Asp 595 600 605 Ser pro Ala Thr Pro Gly Glu Gly lie Leu Ala val val Ser Glu Hi s 610 615 620 cys Ala Arg Gly Ser Leu Hi s Asp Leu Leu Al a Gln Arg Glu ile Lys 625 630 635 640 Leu Asp Trp Met Phe Lys Ser Ser Leu Leu Leu Asp Leu ile Lys Gly 645 650 655 Met Arg Tyr Leu Hi s Hi s Arg Gly Val Ala Hi s Gly Arg Leu Lys Ser 660 665 670 Arg Asn cys Val val Asp Gly Arg Phe val Leu Lys Val Thr Asp Hi s

    675 680 685

    2018203034 01 May 2018

    Gly Hi s 690 Gly Arg Leu Leu Glu Ala Gln Arg val 695 Leu 700 Pro Glu Pro Pro Ser Ala Glu Asp Gln Leu Trp Thr Ala Pro Glu Leu Leu Arg Asp Pro 705 710 715 720 Ser Leu Glu Arg Arg Gly Thr Leu Ala Gly Asp val Phe Ser Leu Ala 725 730 735 lie lie Met Gln Glu Val Val Cys Arg Ser Thr Pro Tyr Ala Met Leu 740 745 750 Glu Leu Thr Pro Glu Glu val lie Gln Arg val Arg Ser Pro Pro Pro 755 760 765 Leu cys Arg Pro Leu val Ser Met Asp Gln Ala Pro Met Glu cys lie 770 775 780 Gln Leu Met Thr Gln cys Trp Ala Glu Hi s Pro Glu Leu Arg Pro Ser 785 790 795 800 Met Asp Leu Thr Phe Asp Leu Phe Lys Ser Ile Asn Lys Gly Arg Lys 805 810 815 Thr Asn lie lie Asp Ser Met Leu Arg Met Leu Glu Gln Tyr Ser Ser 820 825 830 Asn Leu Glu Asp Leu Ile Arg Glu Arg Thr Glu Glu Leu Glu Gln Glu 835 840 845 Lys Gln Lys Thr Asp Arg Leu Leu Thr Gln Met Leu Pro Pro Ser Val 850 855 860 Ala Glu Ala Leu Lys Met Gly Thr Ser val Glu Pro Glu Tyr Phe Glu 865 870 875 880 Glu Val Thr Leu Tyr Phe Ser Asp lie val Gly Phe Thr Thr lie Ser 885 890 895 Ala Met Ser Glu Pro lie Glu val val Asp Leu Leu Asn Asp Leu Tyr 900 905 910 Thr Leu Phe Asp Ala lie Ile Gly Ala Hi s Asp val Tyr Lys val Glu 915 920 925 Thr lie Gly Asp Ala Tyr Met Val Ala Ser Gly Leu Pro Gln Arg Asn 930 935 940 Gly Gln Arg His Ala Al a Glu Ile Ala Asn Met Ser Leu Asp lie Leu 945 950 955 960

    2018203034 01 May 2018

    Ser Ala Val Gly ser Phe Arg Met Arg His Met Pro Glu val Pro val 965 970 975

    Arg lie Arg lie Gly Leu His ser Gly Pro Cys val Ala Gly val val 980 985 990

    Gly Leu Thr Met Pro Arg Tyr Cys Leu Phe Gly Asp Thr val Asn Thr 995 1000 1005

    Ala Ser Arg Met Glu Ser Thr Gly Leu Pro Tyr Arg Ile His Val 1010 1015 1020 Asn Met Ser Thr val Arg Ile Leu Arg Ala Leu Asp Gln Gly Phe 1025 1030 1035 Gln Met Glu cys Arg Gly Arg Thr Glu Leu Lys Gly Lys Gly lie 1040 1045 1050 Glu Asp Thr Tyr Trp Leu val Gly Arg Leu Gly Phe Asn Lys Pro 1055 1060 1065 lie Pro Lys Pro Pro Asp Leu Gln Pro Gly Al a Ser Asn Hi s Gly 1070 1075 1080 Ile Ser Leu Gln Glu Ile Pro Pro Glu Arg Arg Lys Lys Leu Glu 1085 1090 1095 Lys Ala Arg Pro Gly Gln Phe Thr Gly Lys 1100 1105

    <210> 3 <211> 1108 <212> PRT <213> Rattus norvegicus <400> 3

    Met Ser Ala Trp Leu Leu Pro Ala Gly Gly phe Pro Gly Ala Gly Phe 1 5 10 15 cys ile Pro Ala Trp Gln Ser Arg Ser Ser Leu Ser Arg val Leu Arg 20 25 30 Trp Pro Gly Pro Gly Leu Pro Gly Leu Leu Leu Leu Leu Leu Leu Pro 35 40 45 Ser Pro Ser Ala Phe Ser Ala Val Phe Lys val Gly Val Leu Gly Pro 50 55 60 Trp Ala cys Asp Pro lie Phe Ala Arg Ala Arg Pro Asp Leu Ala Al a 65 70 75 80

    Arg Leu Ala Thr Asp Arg Leu Asn Arg Asp Leu Ala Leu Asp Gly Gly 9

    2018203034 01 May 2018

    85 90 95

    Pro Trp Phe Glu Val 100 Thr Leu Leu Pro Glu Pro cys 105 Leu Thr 110 Pro Gly Ser Leu Gly Ala Val Ser Ser Ala Leu Thr Arg val Ser Gly Leu Val 115 120 125 Gly Pro Val Asn Pro Ala Ala cys Arg Pro Ala Glu Leu Leu Ala Gln 130 135 140 Glu Ala Gly Val Al a Leu val pro Trp Gly cys Pro Gly Thr Arg Al a 145 150 155 160 Ala Gly Thr Thr Ala Pro Ala Val Thr Pro Al a Ala Asp Ala Leu Tyr 165 170 175 Val Leu Leu Lys Ala Phe Arg Trp Ala Arg val Ala Leu lie Thr Al a 180 185 190 Pro Gln Asp Leu Trp val Glu Ala Gly Arg Al a Leu Ser Thr Ala Leu 195 200 205 Arg Ala Arg Gly Leu Pro val Ala Leu Val Thr Ser Met val Pro Ser 210 215 220 Asp Leu Ser Gly Ala Arg Glu Ala Leu Arg Arg Ile Arg Asp Gly Pro 225 230 235 240 Arg val Arg val Val ile Met val Met Hi s Ser val Leu Leu Gly Gly 245 250 255 Glu Glu Gln Arg Tyr Leu Leu Glu Ala Al a Glu Glu Leu Gly Leu Thr 260 265 270 Asp Gly Ser Leu Val Phe Leu Pro Phe Asp Thr Leu Hi s Tyr Ala Leu 275 280 285 Ser Pro Gly Pro Glu Ala Leu Ala Ala Phe val Asn Ser ser Lys Leu 290 295 300 Arg Arg Ala Hi s Asp Ala val Leu Thr Leu Thr Arg Arg cys Pro Pro 305 310 315 320 Gly Gly Ser val Gln Asp Ser Leu Arg Arg Ala Gln Glu Hi s Gln Glu 325 330 335 Leu Pro Leu Asp Leu Asp Leu Lys Gln Val Ser Pro Leu Phe Gly Thr 340 345 350 ile Tyr Asp Ala val Phe Leu Leu Ala Gly Gly Val Thr Arg Ala Arg 355 360 365

    2018203034 01 May 2018

    Ala Ala Val 370 Gly Gly Gly Trp val 375 Ser Gly Ala ser 380 Val Ala Arg Gln Met Arg Glu Ala Gln val Phe Gly Phe cys Gly Ile Leu Gly Arg Thr 385 390 395 400 Glu Glu Pro Ser Phe Val Leu Leu Asp Thr Asp Ala Ala Gly Glu Arg 405 410 415 Leu Phe Thr Thr His Leu Leu Asp Pro Val Leu Gly Ser Leu Arg Ser 420 425 430 Ala Gly Thr Pro Val His phe Pro Arg Gly Ala Pro Ala Pro Gly Pro 435 440 445 Asp Pro Ser Cys Trp Phe Asp Pro Asp val ile Cys Asn Gly Gly val 450 455 460 Glu Pro Gly Leu Val Phe Val Gly Phe Leu Leu Val lie Val val Gly 465 470 475 480 Leu Thr Gly Ala phe Leu Ala His Tyr Leu Arg His Arg Leu Leu Hi s 485 490 495 Met Gln Met val Ser Gly Pro Asn Lys Ile Ile Leu Thr Leu Glu Asp 500 505 510 Val Thr Phe Leu His Pro Gln Gly Gly Ser Ser Arg Lys Val Ala Gln 515 520 525 Gly Ser Arg Ser Ser Leu Ala Thr Arg Ser Thr Ser ASp Ile Arg Ser 530 535 540 Val Pro Ser Gln pro Gln Glu Ser Thr Asn lie Gly Leu Tyr Glu Gly 545 550 555 560 ASp Trp Val Trp Leu Lys Lys Phe pro Gly Glu Hi s Hi s Met Ala lie 565 570 575 Arg Pro Ala Thr Lys Met Ala Phe Ser Lys Leu Arg Glu Leu Arg Hi s 580 585 590 Glu Asn val Ala Leu Tyr Leu Gly Leu Phe Leu Ala Gly Thr Ala Asp 595 600 605 ser Pro Ala Thr pro Gly Glu Gly lie Leu Al a val val Ser Glu Hi s 610 615 620 cys Ala Arg Gly Ser Leu Hi s Asp Leu Leu Ala Gln Arg Asp lie Lys 625 630 635 640

    2018203034 01 May 2018

    Leu Asp Trp Met Phe 645 Lys Ser Ser Leu Leu 650 Leu Asp Leu Ile Lys 655 Gly Met Arg Tyr Leu His His Arg Gly val Ala Hi s Gly Arg Leu Lys Ser 660 665 670 Arg Asn cys val val Asp Gly Arg Phe Val Leu Lys val Thr Asp Hi s 675 680 685 Gly Hi s Gly Arg Leu Leu Glu Ala Gin Arg Val Leu Pro Glu Pro Pro 690 695 700 Ser Ala Glu Asp Gin Leu Trp Thr Ala Pro Glu Leu Leu Arg Asp Pro 705 710 715 720 Ala Leu Glu Arg Arg Gly Thr Leu Ala Gly Asp Val Phe Ser Leu Gly 725 730 735 lie lie Met Gin Glu val Val cys Arg Ser Thr Pro Tyr Ala Met Leu 740 745 750 Glu Leu Thr Pro Glu Glu val Ile Gin Arg val Arg Ser pro Pro Pro 755 760 765 Leu cys Arg pro Leu val ser Met Asp Gin Al a Pro Met Glu cys Ile 770 775 780 Gin Leu Met Ala Gin cys Trp Ala Glu Hi s Pro Glu Leu Arg Pro Ser 785 790 795 800 Met Asp Leu Thr Phe Asp Leu Phe Lys Gly lie Asn Lys Gly Arg Lys 805 810 815 Thr Asn lie lie Asp ser Met Leu Arg Met Leu Glu Gin Tyr Ser Ser 820 825 830 Asn Leu Glu Asp Leu lie Arg Glu Arg Thr Glu Glu Leu Glu Gin Glu 835 840 845 Lys Gin Lys Thr Asp Arg Leu Leu Thr Gin Met Leu Pro Pro Ser val 850 855 860 Al a Glu Ala Leu Lys Met Gly Thr Ser val Glu pro Glu Tyr Phe Glu 865 870 875 880 Glu val Thr Leu Tyr Phe Ser Asp ile val Gly Phe Thr Thr Ile Ser 885 890 895 Ala Met ser Glu Pro lie Glu val val Asp Leu Leu Asn Asp Leu Tyr 900 905 910

    2018203034 01 May 2018

    Thr Leu Phe Asp Ala ile Ile Gly Ser His Asp val Tyr Lys val Glu 915 920 925 Thr ile Gly Asp Ala Tyr Met val Ala ser Gly Leu Pro Gln Arg Asn 930 935 940 Gly Gln Arg His Ala Ala Glu ile Ala Asn Met ser Leu Asp Ile Leu 945 950 955 960 Ser Ala Val Gly Ser Phe Arg Met Arg His Met Pro Glu val Pro Val 965 970 975 Arg Ile Arg lie Gly Leu His ser Gly Pro cys val Ala Gly val Val 980 985 990 Gly Leu Thr Met Pro Arg Tyr Cys Leu Phe Gly Asp Thr Val Asn Th 995 1000 1005 Ala Ser Arg Met Glu Ser Thr Gly Leu Pro Tyr Arg ile His val 1010 1015 1020 Asn Met Ser Thr Val Arg Ile Leu Arg Ala Leu Asp Gln Gly Phe 1025 1030 1035 Gln Met Glu Cys Arg Gly Arg Thr Glu Leu Lys Gly Lys Gly val 1040 1045 1050 Glu Asp Thr Tyr Trp Leu Val Gly Arg val Gly Phe Asn Lys Pro 1055 1060 1065 Ile Pro Lys Pro Pro Asp Leu Gln Pro Gly Ala ser Asn His Gly 1070 1075 1080 lie Ser Leu Gln Glu ile Pro Pro Glu Arg Arg Lys Lys Leu Glu 1085 1090 1095 Lys Ala Arg Pro Gly Gln Phe Thr Gly Lys

    1100 1105 <210> 4 <211> 1110 <212> PRT <213> Bos taurus <400> 4

    Met Thr Ala 1 Cys Thr 5 Phe Leu Ala Gly Gly 10 Leu Arg Asp Pro Gly 15 Leu cys Ala Pro Thr Arg Trp Ser Pro Ser Pro Pro Gly Leu Pro pro lie 20 25 30 Pro Pro Arg Pro Arg Leu Arg Leu Arg Pro Pro Leu Leu Leu Leu Leu 35 40 45

    2018203034 01 May 2018

    Leu Leu 50

    Gly Pro 65

    Ala Ala

    Gly Gly

    Pro Gly

    Leu Val 130

    Ala Gln 145

    Arg Ala

    Leu Tyr

    Thr Ala

    Ala Leu 210

    Pro ser 225

    Gly Pro

    Gly Gly

    Leu Ala

    Ala Leu 290

    Gln Leu 305

    Pro

    Trp

    Arg

    Pro

    Ser

    115

    Gly

    Glu

    Ala

    Ala

    Pro

    195

    Arg

    Asp

    Arg

    Glu

    Asp

    275

    Ser

    Arg

    Arg Ser

    Ala cys

    Leu Ala 85

    Arg Phe 100

    Leu Gly

    Pro val

    Ala Gly

    Gly Thr 165

    Leu Leu 180

    Gln Asp

    Ala Arg

    Leu Ser

    Val Arg 245

    Glu Gln 260

    Gly Ser pro Gly

    Lys Ala

    Val

    Asp

    Ala

    Glu

    Ala

    Asn val

    150

    Thr

    Arg

    Leu

    Gly

    Gly

    230

    Ala

    Arg

    Leu

    Pro

    Hi s 310

    Leu Ser 55

    Pro lie

    Ser Arg val Ala

    Val Ser 120 pro Ala 135

    Ala Leu

    Ala Pro

    Ala Phe

    Trp val 200

    Leu Pro 215

    Ala Arg val Ile

    Cys Leu val Phe 280

    Asp Ala 295

    Asp Ala

    Ala Val

    Phe Ala

    Leu Asn 90

    Leu Leu 105

    Ser Ala

    Ala Cys val pro val val 170

    Arg Trp 185

    Glu Ala

    Val Ala

    Glu Ala

    Met val 250

    Leu Glu 265

    Leu Pro

    Leu Ala val Leu

    Phe

    Arg

    His

    Pro

    Leu

    Arg

    Trp

    155

    Thr

    Ala

    Gly

    Leu

    Leu

    235

    Met

    Ala

    Phe val

    Thr

    315

    Thr val 60

    Ala Arg

    Ala Ala

    Glu Pro

    Thr Arg 125

    Pro Ala 140

    Gly Cys

    Pro Ala

    His Val

    His Ala 205 val Thr 220

    Arg Arg

    His ser

    Ala Glu

    Asp Thr 285

    Leu Ala 300

    Leu Thr

    Gly val Leu

    Pro Asp Leu 80

    Ala Leu Glu 95

    Cys Arg Thr 110 val Ser Gly

    Glu Leu Leu

    Pro Gly Thr 160

    Ala Asp Ala 175

    Ala Leu val 190

    Leu Ser Thr

    Ser Met Glu

    Val Gln Asp 240

    Val Leu Leu 255

    Glu Leu Gly 270

    Leu His Tyr

    Asn Ser Ser

    Arg His cys 320

    2018203034 01 May 2018

    Pro Leu Gly Gly Ser 325 val Arg Asp Ser Leu 330 Arg Arg Ala Gln Glu 335 Hi s Arg Glu Leu Pro Leu Asp Leu Asn Leu Gln Gln val Ser Pro Leu Phe 340 345 350 Gly Thr lie Tyr Asp Ser val Phe Leu Leu Ala Gly Gly Val Ala Arg 355 360 365 Ala Arg Val Ala Ala Gly Gly Gly Trp val Ser Gly Ala Ala Val Ala 370 375 380 Arg His lie Arg Asp Ala Arg val Pro Gly Phe cys Gly Ala Leu Gly 385 390 395 400 Gly Ala Glu Glu Pro Ser Phe val Leu Leu Asp Thr Asp Ala Thr Gly 405 410 415 Asp Gln Leu Phe Ala Thr Tyr val Leu Asp Pro Thr Gln Gly Phe Phe 420 425 430 His Ser Ala Gly Thr Pro val Hi s Phe Pro Lys Gly Gly Arg Gly Pro 435 440 445 Gly Pro Asp Pro Ser cys Trp Phe Asp Pro Asp Thr Ile cys Asn Gly 450 455 460 Gly Val Glu Pro ser val Val Phe lie Gly Phe Leu Leu Val val Gly 465 470 475 480 Met Gly Leu Ala Gly Ala Phe Leu Ala His Tyr Cys Arg Hi s Arg Leu 485 490 495 Leu His lie Gln Met val Ser Gly Pro Asn Lys lie Ile Leu Thr Leu 500 505 510 Asp Asp lie Thr Phe Leu Hi s Pro Hi s Gly Gly Asn ser Arg Lys Val 515 520 525 Al a Gln Gly Ser Arg Thr Ser Leu Ala Al a Arg Ser Ile Ser Asp val 530 535 540 Arg Ser Ile Hi s Ser Gln Leu Pro Asp Tyr Thr Asn Ile Gly Leu Tyr 545 550 555 560 Glu Gly Asp Trp Val Trp Leu Lys Lys Phe Pro Gly Asp Arg Hi s Ile 565 570 575 Ala lie Arg Pro Ala Thr Lys Met Ala Phe ser Lys ile Arg Glu Leu 580 585 590

    2018203034 01 May 2018

    Arg His

    Ala Gly 610

    Glu His 625

    Ile Lys

    Lys Gly

    Lys Ser

    Asp His 690

    Pro Pro 705

    Asp Pro

    Leu Gly

    Met Leu

    Pro Pro 770

    Cys Ile 785

    Pro Ser

    Arg Lys

    Ser Ser

    Leu Glu 850

    Ser val

    Glu Asn 595

    Gly Pro

    Cys Ala

    Leu Asp

    Ile Arg 660

    Arg Asn 675

    Gly His

    Ser Ala

    Val Leu lie lie 740

    Glu Leu 755

    Leu Cys

    Gln Leu

    Met Asp

    Met Asn 820

    Asn Leu 835

    Lys Gln

    Ala Glu

    Val

    Ala

    Arg

    Trp

    645

    Tyr cys

    Gly

    Glu

    Glu

    725

    Met

    Thr

    Arg

    Met

    Arg

    805

    Ile

    Glu

    Lys

    Ala

    Ala Leu

    Ala Pro 615

    Gly Ser 630

    Met Phe

    Leu His

    Val val

    Arg Leu 695

    Asp Gln 710

    Arg Arg

    Gln Glu

    Pro Glu

    Pro Ser 775

    Lys Gln 790

    Thr Phe lie Asp

    Asp Leu

    Thr Asp 855

    Leu Lys

    Tyr Leu 600

    Gly Glu

    Leu Gln

    Lys Ser

    His Arg 665

    Asp Gly 680

    Leu Glu

    Leu Trp

    Gly Thr

    Val val 745

    Glu val 760 val Ser

    Cys Trp

    Glu Leu ser Met 825 ile Arg 840

    Arg Leu

    Met Gly

    Gly Leu

    Gly val

    Asp Leu 635

    Ser Leu 650

    Gly val

    Arg Phe

    Ala Gln

    Thr Ala 715

    Leu Ala 730

    Cys Arg val Lys ile Asp

    Ala Glu 795

    Phe Lys 810

    Leu Arg

    Glu Arg

    Leu Thr

    Thr Pro 16

    Phe Leu 605

    Leu Ala 620

    Leu Ala

    Leu Leu

    Ala His val Leu 685

    Arg val 700

    Pro Glu

    Gly Asp

    Ser Ala

    Arg val 765

    Gln Ala 780

    Gln Pro

    Ser lie

    Met Leu

    Thr Glu 845

    Gln Met 860 val Glu

    Ala

    Val

    Gln

    Asp

    Gly

    670

    Lys

    Leu

    Leu val

    Pro

    750

    Gln

    Pro

    Glu

    Asn

    Glu

    830

    Glu

    Leu

    Pro

    Gly Gly

    Val Ser

    Arg Asp 640

    Leu lie 655

    Arg Leu

    Val Thr

    Pro Glu

    Leu Arg 720

    Phe Ser 735

    Tyr Ala

    Ser Pro

    Met Glu

    Leu Arg 800

    Lys Gly 815

    Gln Tyr

    Leu Glu

    Pro Pro

    Glu Tyr

    2018203034 01 May 2018

    865

    870

    875

    880

    Phe Glu Glu val Thr Leu Tyr Phe Ser 885 Asp lie 890 Val Gly Phe Thr 895 Thr lie Ser Ala Met Ser Glu Pro Ile Glu val val Asp Leu Leu Asn Asp 900 905 910 Leu Tyr Thr Leu Phe Asp Ala Ile Ile Gly Ser His Asp val Tyr Lys 915 920 925 val Glu Thr ile Gly Asp Ala Tyr Met val Al a Ser Gly Leu Pro Gln 930 935 940 Arg Asn Gly Hi s Arg Hi s Ala Ala Glu Ile Ala Asn Met Ala Leu Asp 945 950 955 960 Ile Leu Ser Ala val Gly Thr Phe Arg Met Arg Hi s Met Pro Glu Val 965 970 975 Pro Val Arg lie Arg lie Gly Leu Hi s Ser Gly Pro cys val Ala Gly 980 985 990

    Val val Gly Leu Thr Met Pro Arg Tyr cys Leu Phe Gly Asp Thr val 995 1000 1005

    Asn Thr Ala Ser Arg Met Glu 1015 Ser Thr Gly Leu Pro 1020 Tyr Arg ile 1010 Hi s Val Asn Arg ser Thr val Gln ile Leu Ser Ala Leu Asn Glu 1025 1030 1035 Gly Phe Leu Thr Glu val Arg Gly Arg Thr Glu Leu Lys Gly Lys 1040 1045 1050 Gly Ala Glu Glu Thr Tyr Trp Leu val Gly Arg Arg Gly Phe Asn 1055 1060 1065 Lys Pro ile Pro Lys Pro Pro Asp Leu Gln Pro Gly Al a Ser Asn 1070 1075 1080 Hi s Gly lie Ser Leu His Glu lie Pro Pro Asp Arg Arg Gln Lys 1085 1090 1095 Leu Glu Lys Ala Arg pro Gly Gln Phe ser Gly Lys 1100 1105 1110

    <210> 5 <211> 1109 <212> PRT <213> Canis f ami Haris <400> 5

    2018203034 01 May 2018

    Met 1 Ser Ala cys Ala Leu Leu Ala Gly 5 Gly Leu 10 Pro Asp Pro Arg 15 Leu cys Ala Pro Ala Arg Trp Ala Arg Ser Pro Pro Gly val Pro Gly Ala 20 25 30 Pro Pro Trp Pro Gln Pro Arg Leu Arg Leu Leu Leu Leu Leu Leu Leu 35 40 45 Leu Pro Pro Ser Ala Leu ser Ala Val Phe Thr Val Gly Val Leu Gly 50 55 60 Pro Trp Ala cys Asp Pro lie Phe Ala Arg Ala Arg Pro Asp Leu Ala 65 70 75 80 Ala Arg Leu Ala Ala Ala Arg Leu Asn Arg Asp Ala Ala Leu Glu Asp 85 90 95 Gly Pro Arg Phe Glu val Thr Leu Leu Pro Glu Pro cys Arg Thr Pro 100 105 110 Gly Ser Leu Gly Ala val Ser Ser Ala Leu Gly Arg val Ser Gly Leu 115 120 125 val Gly Pro val Asn Pro Ala Ala cys Arg Pro Ala Glu Leu Leu Ala 130 135 140 Gln Glu Ala Gly val Ala Leu val Pro Trp Ser cys Pro Gly Thr Arg 145 150 155 160 Ala Gly Gly Thr Thr Ala Pro Ala Gly Thr Pro Ala Ala Asp Ala Leu 165 170 175 Tyr Ala Leu Leu Arg Ala Phe Arg Trp Ala Arg val Ala Leu lie Thr 180 185 190 Ala Pro Gln Asp Leu Trp val Glu Ala Gly Arg Ala Leu Ser Ala Ala 195 200 205 Leu Arg Ala Arg Gly Leu Pro Val Ala Leu val Thr Thr Met Glu Pro 210 215 220 ser Asp Leu ser Gly Al a Arg Glu Ala Leu Arg Arg val Gln Asp Gly 225 230 235 240 Pro Arg Val Arg Ala Val Ile Met val Met Hi s Ser val Leu Leu Gly 245 250 255 Gly Glu Glu Gln Arg cys Leu Leu Gln Al a Ala Glu Glu Leu Gly Leu 260 265 270

    2018203034 01 May 2018

    Ala Asp Gly Ser Leu val Phe Leu Pro Phe Asp Thr Leu Hi s Tyr Ala 275 280 285 Leu Ser Pro Gly Pro Glu Ala Leu Ala Val Leu Ala Asn Ser Ser Gln 290 295 300 Leu Arg Arg Ala Hl s Asp Ala val Leu lie Leu Thr Arg Hi s cys Pro 305 310 315 320 Pro Gly Gly Ser val Met Asp Asn Leu Arg Arg Ala Gln Glu Hi s Gln 325 330 335 Glu Leu Pro Ser Asp Leu Asp Leu Gln Gln Val Ser Pro Phe Phe Gly 340 345 350 Thr Ile Tyr Asp Ala Val Leu Leu Leu Ala Gly Gly val Ala Arg Al a 355 360 365 Arg Ala Ala Ala Gly Gly Gly Trp val Ser Gly Ala Thr val Ala His 370 375 380 Hl s Ile Pro Asp Ala Gln val Pro Gly Phe Cys Gly Thr Leu Gly Gly 385 390 395 400 Ala Gln Glu Pro pro Phe val Leu Leu Asp Thr Asp Ala Ala Gly Asp 405 410 415 Arg Leu Phe Ala Thr Tyr Met Leu Asp Pro Thr Arg Gly Ser Leu Leu 420 425 430 Ser Ala Gly Thr Pro val His Phe Pro Arg Gly Gly Gly Thr Pro Gly 435 440 445 Ser Asp Pro Ser Cys Trp Phe Glu pro Gly val lie cys Asn Gly Gly 450 455 460 val Glu Pro Gly Leu val Phe Leu Gly Phe Leu Leu val val Gly Met 465 470 475 480 Gly Leu Thr Gly Ala Phe Leu Ala Hi s Tyr Leu Arg Hi s Arg Leu Leu 485 490 495 Hi s lie Gln Met Val ser Gly Pro Asn Lys Ile Ile Leu Thr Leu Asp 500 505 510 Asp val Thr Phe Leu Hi s Pro Hl s Gly Gly Ser Thr Arg Lys Val val 515 520 525 Gln Gly Ser Arg ser Ser Leu Ala Ala Arg Ser Thr Ser Asp Ile Arg 530 535 540 Ser Val Pro Ser Gln Pro Leu Asp Asn Ser Asn Ile Gly Leu Phe Glu 19

    2018203034 01 May 2018

    545 550

    Gly Asp Trp Val Trp 565 Leu Lys Lys lie Arg Pro Ala 580 Thr Lys Thr Ala Hi s Glu Asn 595 Val Val Leu Tyr Leu 600 Ala Gly 610 Gly Ser Ala Ala Gly 615 Glu Hi s 625 Cys Ala Arg Gly Ser 630 Leu Hi s Lys Leu Asp Trp Met 645 Phe Lys Ser Gly Met Arg Tyr 660 Leu Hi s His Arg Ser Arg Asn 675 cys val val Asp Gly 680 His Gly 690 Hi s Ala Arg Leu Met 695 Glu Pro 705 Ser Ala Glu Asp Gin 710 Leu Trp Pro Ala Leu Glu Arg 725 Arg Gly Thr Gly lie lie Met 740 Gin Glu val val Leu Glu Leu 755 Thr Pro Glu Glu Val 760 Pro Leu 770 cys Arg Pro Ser val 775 Ser lie 785 Gin Leu Met Lys Gin 790 cys Trp Ser Leu Gly His lie 805 Phe Asp Gin Lys Thr Asn ile 820 Ile Asp ser Met

    555 560 Phe Pro 570 Gly Asp Gin Hi s Ile 575 Al a Phe 585 Ser Lys Leu Arg Glu 590 Leu Arg Gly Leu Phe Leu Gly 605 Ser Gly Gly Gly val Leu Ala 620 Val val Ser Glu Asp Leu Leu 635 Ala Gin Arg Asp Ile 640 Ser Leu 650 Leu Leu Asp Leu lie 655 Lys Gly 665 val Ala Hi s Gly Arg 670 Leu Lys Arg Phe val Leu Lys 685 val Thr Asp Ala Gin Arg val 700 Leu Leu Glu Pro Thr Ala Pro 715 Glu Leu Leu Arg Asp 720 Leu Pro 730 Gly Asp val Phe Ser 735 Leu cys 745 Arg Ser Ala Pro Tyr 750 Ala Met Val Glu Arg val Arg 765 Ser Pro Pro Met Asp Gin Ala 780 Pro Val Glu cys Ala Glu Hi s 795 Pro Asp Leu Arg Pro 800 Phe Lys 810 Ser lie Asn Lys Gly 815 Arg Leu 825 Arg Met Leu Glu Gin 830 Tyr Ser

    2018203034 01 May 2018

    Ser Asn Leu Glu Asp Leu Ile Arg Glu Arg Thr Glu Glu Leu Glu Leu 835 840 845

    Glu Lys Gln 850 Lys Thr Asp Arg Leu 855 Leu Thr Gln Met Leu 860 Pro Pro Ser Val Ala Glu Ala Leu Lys Met Gly Thr Pro Val Glu Pro Glu Tyr Phe 865 870 875 880 Glu Glu val Thr Leu Tyr Phe Ser Asp lie Val Gly Phe Thr Thr Ile 885 890 895 Ser Ala Met Ser Glu Pro lie Glu Val Val Asp Leu Leu Asn Asp Leu 900 905 910 Tyr Thr Leu Phe Asp Ala lie lie Gly Ser Hi s Asp Val Tyr Lys Val 915 920 925 Glu Thr Ile Gly Asp Ala Tyr Met val Ala Ser Gly Leu Pro Gln Arg 930 935 940 Asn Gly Gln Arg Hi s Ala Ala Glu ile Ala Asn Met Ala Leu Asp ile 945 950 955 960 Leu Ser Ala val Gly Ser Phe Arg Met Arg His Met Pro Glu val Pro 965 970 975 val Arg ile Arg ile Gly Leu Hi s Ser Gly Pro cys Val Ala Gly Val 980 985 990

    Val Gly Leu Thr Met Pro Arg Tyr Cys Leu Phe Gly Asp Thr Val Asn 995 1000 1005

    Thr Ala Ser Arg Met Glu Ser Thr Gly Leu Pro Tyr Arg Ile His 1010 1015 1020

    Val Asn Met Ser Thr val Arg Ile Leu His Ala Leu Asp Glu Gly 1025 1030 1035

    Phe Gln Thr Glu val Arg Gly Arg Thr Glu Leu Lys Gly Lys Gly 1040 1045 1050

    Ala Glu Asp Thr Tyr Trp Leu val Gly Arg Arg Gly Phe Asn Lys 1055 1060 1065

    Pro lie Pro Lys Pro Pro Asp Leu Gln Pro Gly Ala ser Asn His 1070 1075 1080

    Gly Ile Ser Leu Gln Glu Ile Pro Leu Asp Arg Arg Trp Lys Leu 1085 1090 1095

    2018203034 01 May 2018

    Glu Lys Ala Arg Pro Gly Gln Phe Ser Gly Lys 1100 1105 <210> 6 <211> 1103 <212> PRT <213> Macaca mulatta <400> 6

    Met 1 Thr Ala cys Ala 5 Arg Arg Ala Gly Gly Leu Pro Asp Pro Arg Leu 10 15 cys Gly Pro Ala Arg Trp Ala Pro Ala Leu Pro Arg Leu Pro Arg Ala 20 25 30 Leu Pro Arg Leu Pro Leu Leu Leu Leu Leu Leu Leu Leu Gln Pro Pro 35 40 45 Ala Leu Ser Ala val Phe Thr val Gly val Leu Gly Pro Trp Ala Cys 50 55 60 Asp Pro ile Phe Ser Arg Ala Arg Ala Asp Leu Ala Ala Arg Leu Ala 65 70 75 80 Ala Ala Arg Leu Asn Arg Asp pro Asp Leu Ala Gly Gly Pro Arg Phe 85 90 95 Glu val Ala Leu Leu Pro Glu Pro cys Arg Thr Pro Gly Ser Leu Gly 100 105 110 Ala val Ser ser Ala Leu Thr Arg Val Ser Gly Leu val Gly Pro val 115 120 125 Asn Pro Ala Ala cys Arg Pro Ala Glu Leu Leu Ala Glu Glu Ala Gly 130 135 140 ile Ala Leu val pro Trp Gly cys Pro Gly Thr Gln Ala Ala Gly Thr 145 150 155 160 Thr Ala Pro Ala Leu Thr Pro Ala Ala Asp Ala Leu Tyr Al a Leu Leu 165 170 175 Arg Ala Phe Gly Trp Ala Arg val Ala Leu Val Thr Ala Pro Gln Asp 180 185 190 Leu Trp val Glu Ala Gly Hi s Ser Leu Ser Thr Ala Leu Arg Al a Arg 195 200 205 Gly Leu Pro val Ala Ser val Thr Ser Met Glu Pro Leu Asp Leu Ser

    210 215 220

    Gly Ala Arg Glu Ala Leu Arg Lys Val Arg Asp Gly Pro Arg Val Thr 22

    2018203034 01 May 2018

    225 230

    Ala val ile Met Val 245 Met Hi s Ser Arg Tyr Leu Leu 260 Glu Ala Ala Glu Leu val Phe 275 Leu Pro Phe Asp Thr 280 Pro Glu 290 Ala Leu Ala Ala Leu 295 Ala Hi s 305 Asp Ala val Leu Thr 310 Leu Thr Val Leu Asp Ser Leu 325 Arg Arg Ala Asp Leu Asn Leu 340 Gln Gln val Ser Ala val Phe 355 Leu Leu Val Arg Gly 360 Gly Gly 370 Arg Trp val Ser Gly 375 Ala Ala 385 Gln Val Pro Gly Phe 390 cys Gly Pro Phe val Leu Leu 405 Asp Thr Asp Thr Tyr Met Leu 420 Asp Pro Thr Arg Pro Met Hi s 435 Phe Pro Arg Gly Gly 440 cys Trp 450 Phe Asp Pro Asn Asn 455 Ile Leu 465 val Phe Leu Gly Phe 470 Leu Leu Ala Phe Leu Ala His 485 Tyr val Arg val Ser Gly pro 500 Asn Lys Ile lie

    235 240

    val Leu 250 Leu Gly Gly Glu Glu 255 Gln Glu 265 Leu Gly Leu Thr Asp 270 Gly Ser Val His Tyr Ala Leu 285 Ser pro Gly Asn Ser Ser Gln 300 Leu Arg Arg Ala Arg His cys 315 Pro Ser Glu Gly Ser 320 Gln Glu 330 Arg Arg Glu Leu Pro 335 Ser Pro 345 Leu Phe Gly Thr lie 350 Tyr Asp Val Ala Glu Ala Arg 365 Ala Ala Ala Ala Val Al a Arg 380 His val Trp Asp Asp Leu Gly 395 Gly Asp Glu Glu Pro 400 Ala val 410 Gly Asp Arg Leu Phe 415 Al a Gly 425 Ser Leu Leu Ser Ala 430 Gly Thr Ser Ala Pro Gly Pro 445 Asp Pro Ser cys Gly Gly Gly 460 Leu Glu pro Gly val Val Gly 475 Met Gly Leu Al a Gly 480 Hi s Gln 490 Leu Leu Hi s Ile Gln 495 Met Leu Thr val Asp Asp ile Thr Phe

    505 510

    2018203034 01 May 2018

    Leu His Pro His 515 Gly Gly Thr Ser 520 Arg Lys val Ala Gln 525 Gly Ser Arg ser Ser Leu Ala Ala Arg Ser Met Ser Asp val Arg Ser Gly Pro Ser 530 535 540 Gln Pro Thr Asp Ser Pro Asn Val Gly val Tyr Glu Gly Asp Arg Val 545 550 555 560 Trp Leu Lys Lys Phe Pro Gly Asp Gln Hi s Ile Ala lie Arg Pro Al a 565 570 575 Thr Lys Thr Ala Phe Ser Lys Leu Gln Glu Leu Arg Hi s Glu Asn Val 580 585 590 Ala Leu Tyr Leu Gly Leu Phe Leu Ala Gln Gly Ala Glu Gly Pro Ala 595 600 605 Ala Leu Trp Glu Gly Asn Leu Ala val Val Ser Glu His Cys Thr Arg 610 615 620 Gly Ser Leu Gln Asp Leu Leu Ala Gln Arg Glu Ile Lys Leu Asp Trp 625 630 635 640 Met Phe Lys ser Ser Leu Leu Leu Asp Leu Ile Lys Gly lie Arg Tyr 645 650 655 Leu Hi s Hi s Arg Gly Val Ala Hi s Gly Arg Leu Lys Ser Arg Asn cys 660 665 670 lie val Asp Gly Arg Phe val Leu Lys lie Thr Asp Hi s Gly Hi s Gly 675 680 685 Arg Leu Leu Glu Ala Gln Lys val Leu Pro Glu Pro Pro Arg Ala Glu 690 695 700 Asp Gln Leu Trp Thr Al a Pro Glu Leu Leu Arg Asp Pro Ala Leu Glu 705 710 715 720 Arg Arg Gly Thr Leu Al a Gly Asp val Phe Ser Leu Ala lie lie Met 725 730 735 Gln Glu Val val cys Arg Ser Ala Pro Tyr Al a Met Leu Glu Leu Thr 740 745 750 Pro Glu Glu Val val Gln Arg val Arg ser Pro pro pro Leu Cys Arg 755 760 765 Pro Leu val Ser Met Asp Gln Ala Pro val Glu cys Ile Hi s Leu Met

    770 775 780

    2018203034 01 May 2018

    Lys 785 Gln cys Trp Ala Glu 790 Gln Pro Glu Leu Arg 795 Pro Ser Met Asp Hi s 800 Thr Phe Asp Leu Phe Lys Asn He Asn Lys Gly Arg Lys Thr Asn Ile 805 810 815 Ile Asp Ser Met Leu Arg Met Leu Glu Gln Tyr Ser Ser Asn Leu Glu 820 825 830 Asp Leu Ile Arg Glu Arg Thr Glu Glu Leu Glu Leu Glu Lys Gln Lys 835 840 845 Thr Asp Arg Leu Leu Thr Gln Met Leu Pro Pro Ser Val Ala Glu Ala 850 855 860 Leu Lys Thr Gly Thr Pro Val Glu Pro Glu Tyr Phe Glu Gln Val Thr 865 870 875 880 Leu Tyr Phe Ser Asp Ile Val Gly Phe Thr Thr Ile Ser Ala Met Ser 885 890 895 Glu Pro lie Glu val val Asp Leu Leu Asn Asp Leu Tyr Thr Leu Phe 900 905 910 Asp Ala lie ile Gly Ser Hi s Asp val Tyr Lys val Glu Thr lie Gly 915 920 925 Asp Ala Tyr Met val Al a Ser Gly Leu Pro Gln Arg Asn Gly Gln Arg 930 935 940 His Ala Ala Glu lie Ala Asn Met Ser Leu Asp Ile Leu Ser Ala val 945 950 955 960 Gly Thr Phe Arg Met Arg His Met Pro Glu val Pro Val Arg lie Arg 965 970 975 Ile Gly Leu His Ser Gly Pro cys Val Ala Gly Val val Gly Leu Thr 980 985 990

    Met

    Met

    Thr

    Leu

    Pro Arg Tyr Cys Leu 995

    Phe Gly Asp Thr Val Asn Thr Ala Ser Arg 1000 1005

    Glu 1010 Ser Thr Gly Leu Pro 1015 Tyr Arg ile Hi s val 1020 Asn Leu Ser Val Gly ile Leu Arg Ala Leu Asp Ser Gly Tyr Gln Val Glu 1025 1030 1035 Arg Gly Arg Thr Glu Leu Lys Gly Lys Gly Ala Glu Asp Thr 1040 1045 1050

    2018203034 01 May 2018

    Phe Trp 1055 Leu val Gly Arg Arg 1060 Gly Phe Asn Lys Pro 1065 Ile Pro Pro Pro 1070 Asp Leu Gln Pro Gly 1075 Ser ser Asn Hi s Gly 1080 Ile Ser Gln Glu 1085 Ile Pro pro Glu Arg 1090 Arg Arg Lys Leu Glu 1095 Lys Ala Pro Gly 1100 Gln Phe Ser

    Lys

    Leu

    Arg <210> 7 <211> 1103 <212> PRT <213> Pongo pygmaeus <400> 7

    Met 1 Thr Ala cys Ala 5 Arg Arg Ala cys Gly pro Ala 20 Arg Trp Ala Pro Leu Pro Arg 35 Leu Pro Leu Leu Leu 40 Ala Leu 50 ser Ala val Phe Thr 55 val Asp 65 Pro lie Phe Ser Arg 70 Ala Arg Ala Ala Arg Leu Asn 85 Arg Asp Pro Glu val Ala Leu 100 Leu Pro Glu Pro Ala val Ser 115 Ser Ala Leu Ala Arg 120 Asn Pro 130 Ala Ala cys Arg Pro 135 Ala Ile 145 Ala Leu val Pro T rp 150 Gly Cys Thr Ala Pro Cys val 165 Thr Pro Ala Arg Ala Phe Gly 180 Trp Ala Arg Val

    Gly Gly 10 Leu Pro Asp Pro Gly 15 Leu Ser 25 Leu Pro Arg Leu Pro 30 Arg Al a Leu Leu Leu Leu Leu 45 Gln Pro Pro Gly val Leu Gly 60 Pro Trp Al a Cys Pro Asp Leu 75 Ala Al a Arg Leu Ala 80 Gly Leu 90 Al a Gly Gly Pro Arg 95 Phe Cys 105 Arg Thr Pro Gly Ser 110 Leu Gly Val Ser Gly Leu val 125 Gly Pro Val Glu Leu Leu Ala 140 Asp Asn pro Gly Pro Trp Thr 155 Gln Ala Glu Gly Thr 160 Ala Asp 170 Ala Leu Tyr Ala Leu 175 Leu Ala Leu val Thr Ala Pro Gln Asp

    185 190

    2018203034 01 May 2018

    Leu Trp Val Glu Ala Gly Arg Ser Leu Ser Thr Ala Leu Arg Ala Arg 195 200 205 Gly Leu Pro val Ala ser val Thr Ser Met Glu Pro Leu Asp Leu Ser 210 215 220 Gly Ala Arg Glu Ala Leu Arg Lys Val Arg Asp Gly Pro Arg val Thr 225 230 235 240 Ala val ile Met Val Met Hi s ser Val Leu Leu Gly Gly Glu Glu Gin 245 250 255 Arg Tyr Leu Leu Glu Ala Ala Glu Glu Leu Gly Leu Thr Asp Gly Ser 260 265 270 Leu val Phe Leu Pro Phe Asp Thr Ile Hi s Tyr Ala Leu Ser Pro Gly 275 280 285 Pro Glu Ala Leu Ala Ala Leu Ala Asn Ser Ser Gin Leu Arg Arg Ala 290 295 300 His Asp Ala Val Leu Thr Leu Thr Arg His Cys Pro Ser Glu Gly Ser 305 310 315 320 Val Leu Asp ser Leu Arg Arg Ala Gin Glu Arg Arg Glu Leu Pro Ser 325 330 335 Asp Leu Asn Leu Gin Gl n Val Ser Pro Leu Phe Gly Thr lie Tyr Asp 340 345 350 Ala val Phe Leu Leu Ala Arg Gly Val Ala Glu Al a Trp Ala Ala Al a 355 360 365 Gly Gly Arg Trp Val Ser Gly Ala Ala val Ala Arg His lie Arg Asp 370 375 380 Ala Gin val Pro Gly Phe cys Gly Asp Leu Gly Gly Asp Gly Glu Pro 385 390 395 400 Pro Phe val Leu Leu Asp Thr Asp Ala Ala Gly Asp Arg Leu Phe Al a 405 410 415 Thr Tyr Met Leu Asp Pro Ala Arg Gly ser Phe Leu ser Ala Gly Thr 420 425 430 Arg Met His Phe pro Arg Gly Gly Ser Ala Pro Gly Pro Asp Pro Ser 435 440 445 Cys Trp Phe Asp Pro Asn Asn Ile Cys Gly Gly Gly Leu Glu Pro Gly 450 455 460

    2018203034 01 May 2018

    Leu val 465 Phe Leu Gly Phe 470 Leu Leu Val val Gly Met 475 Gly Leu Ala Gly 480 Ala Phe Leu Ala His Tyr val Arg Hi s Arg Leu Leu His lie Gln Met 485 490 495 val Ser Gly Pro Asn Lys lie Ile Leu Thr Val Asn Asp lie Thr Phe 500 505 510 Leu Hi s Pro Hi s Gly Gly Thr Ser Arg Lys Val Ala Gln Gly Ser Arg 515 520 525 Ser Ser Leu Ala Ala Arg Ser Met Ser Asp lie Arg Ser Gly Pro Ser 530 535 540 Gln Pro Leu Asp ser Pro Asn val Gly val Tyr Glu Gly Asp Arg val 545 550 555 560 Trp Leu Lys Lys Phe Pro Gly Asp Gln His Ile Ala Ile Arg Pro Al a 565 570 575 Thr Lys Thr Ala Phe Ser Lys Leu Gln Glu Leu Arg Hi s Glu Asn val 580 585 590 Ala Leu Tyr Leu Gly Leu Phe Leu Ala Arg Gly Al a Glu Gly Pro Al a 595 600 605 Ala Leu Trp Glu Gly Asn Leu Ala Val val Ser Glu Hi s Cys Thr Arg 610 615 620 Gly Ser Leu Gln Asp Leu Leu Ser Gln Arg Glu Ile Lys Leu Asp T rp 625 630 635 640 Met Phe Lys Ser Ser Leu Leu Leu Asp Leu Ile Lys Gly ile Arg Tyr 645 650 655 Leu Hi s Hi s Arg Gly val Ala Hi s Gly Arg Leu Lys Ser Arg Asn cys 660 665 670 ile val Asp Gly Arg Phe val Leu Lys Ile Thr Asp His Gly His Gly 675 680 685 Arg Leu Leu Glu Ala Gln Lys val Leu Pro Glu Pro Pro Arg Al a Glu 690 695 700 Asp Gln Leu Trp Thr Ala Pro Glu Leu Leu Arg Asp Pro Ala Leu Glu 705 710 715 720 Arg Arg Gly Thr Leu Ala Gly Asp val Phe Ser Leu Ala Ile lie Met 725 730 735

    2018203034 01 May 2018

    Gln Glu Val val 740 cys Arg Ser Ala Pro 745 Tyr Ala Met Leu Glu 750 Leu Thr Pro Glu Glu val val Gln Arg Val Arg Ser Pro Pro pro Leu cys Arg 755 760 765 Pro Leu val Ser Met Asp Gln Ala Pro val Glu cys Ile Hi s Leu Met 770 775 780 Lys Gln Cys Trp Ala Glu Gln Pro Glu Leu Arg Pro Ser Met Asp Hi s 785 790 795 800 Thr Phe Asp Leu Phe Lys Asn Ile Asn Lys Gly Arg Lys Thr Asn ile 805 810 815 lie Asp Ser Met Leu Arg Met Leu Glu Gln Tyr ser ser Asn Leu Glu 820 825 830 Asp Leu Ile Arg Glu Arg Thr Glu Glu Leu Glu Leu Glu Lys Gln Lys 835 840 845 Thr Asp Arg Leu Leu Thr Gln Met Leu Pro Pro ser val Ala Glu Al a 850 855 860 Leu Lys Thr Gly Thr Pro val Glu Pro Glu Tyr Phe Glu Gln val Thr 865 870 875 880 Leu Tyr Phe Ser Asp Ile val Gly Phe Thr Thr lie ser Ala Met Ser 885 890 895 Glu Pro lie Glu Val val Asp Leu Leu Asn Asp Leu Tyr Thr Leu Phe 900 905 910 Asp Ala lie ile Gly Ser His Asp Val Tyr Lys Val Glu Thr Ile Gly 915 920 925 Asp Ala Tyr Met val Ala Ser Gly Leu Pro Gln Arg Asn Gly Gln Arg 930 935 940 Hi s Ala Ala Glu lie Ala Asn Met Ser Leu Asp Ile Leu Ser Ala val 945 950 955 960 Gly Thr Phe Arg Met Arg His Met Pro Glu val Pro val Arg lie Arg 965 970 975 lie Gly Leu His Ser Gly Pro Cys Val Ala Gly Val val Gly Leu Thr 980 985 990 Met Pro Arg Tyr Cys Leu Phe Gly Asp Thr Val Asn Thr Ala Ser Arg 995 1000 1005

    Met Glu Ser Thr Gly Leu Pro Tyr Arg lie His val Asn Leu Ser 29

    2018203034 01 May 2018

    1010 1015 1020 Thr val Gly lie Leu Arg Ala Leu Asp Ser Gly Tyr Gln val Glu 1025 1030 1035 Leu Arg Gly Arg Thr Glu Leu Lys Gly Lys Gly Ala Glu Asp Thr 1040 1045 1050 Phe Trp Leu Val Gly Arg Arg Gly Phe Asn Lys Pro Ile Pro Lys 1055 1060 1065 Pro Pro Asp Leu Gln Pro Gly Ser Ser Asn His Gly Ile ser Leu 1070 1075 1080 Gln Glu Ile Pro Pro Glu Arg Arg Arg Lys Leu Glu Lys Ala Arg 1085 1090 1095

    Pro Gly Gln Phe Ser 1100 <210> 8 <211> 1103 <212> PRT <213> Callithrix jacchus <400> 8

    Met 1 Thr Ala Cys Ala Arg 5 Arg Ala Gly Gly 10 Leu Pro Asp Pro Gly 15 Leu cys Gly Pro Ala Arg Trp Ala Pro Ala Leu Ser Arg Leu Pro Arg Ala 20 25 30 Leu Pro Arg Leu Pro Leu Leu Leu Leu Leu Leu Leu Leu Gln Pro Pro 35 40 45 Ala Leu Ser Ala Gln Phe Thr val Gly val Leu Gly Pro Trp Ala Cys 50 55 60 Asp Pro lie Phe Ser Arg Ala Arg Pro Asp Leu Ala Ala Arg Leu Al a 65 70 75 80 Ala Ala Arg Leu Asn Arg Asp Pro Ser Leu Ala Gly Gly Pro Arg Phe 85 90 95 Glu val Al a Leu Leu Pro Glu Pro cys Arg Thr Pro Gly Ser Leu Gly 100 105 110 Al a val Ser Ser Ala Leu Ala Arg val Ser Gly Leu val Gly pro val 115 120 125 Asn Pro Ala Ala Cys Arg Pro Ala Glu Leu Leu Al a Glu Glu Ala Gly

    130 135 140

    2018203034 01 May 2018

    lie 145 Ala Leu val Pro Trp Gly Cys Pro 150 Gly Thr Gln 155 Ala Ala Gly Thr 160 Thr Ala Pro Val Val Thr Pro Ala Ala Asp Ala Leu Tyr Ala Leu Leu 165 170 175 Arg Ala Phe Gly Trp Al a Arg val Ala Leu Val Thr Ala Pro Gln Asp 180 185 190 Leu Trp val Glu Ala Gly Leu Ser Leu Ser Thr Ala Leu Arg Ala Arg 195 200 205 Gly Leu Pro val val Ser val Thr Ser Met Glu Pro Leu Asp Leu ser 210 215 220 Gly Ala Arg Glu Ala Leu Arg Lys val Arg Asn Gly Pro Arg val Thr 225 230 235 240 Ala val lie Met val Met Hi s Ser val Leu Leu Gly Gly Glu Glu Gln 245 250 255 Arg Tyr Leu Leu Glu Ala Ala Glu Glu Leu Gly Leu Thr Asp Gly Ser 260 265 270 Leu val Phe Leu Pro Phe Asp Thr Ile Hi s Tyr Ala Leu Ser Pro Gly 275 280 285 Arg Glu Ala Leu Ala Ala Leu Val Asn Ser Ser Gln Leu Arg Arg Ala 290 295 300 Hi s Asp Ala val Leu Thr Leu Thr Arg His Cys Ser Ser Glu Gly Ser 305 310 315 320 val Leu Asp Ser Leu Arg Lys Ala Gln Gln Arg Arg Glu Leu Pro Ser 325 330 335 Asp Leu Asn Leu Glu Gln Val ser Pro Leu Phe Gly Thr lie Tyr Asp 340 345 350 Ala val val Leu Leu Ala Arg Gly Val Ala Asp Ala Arg Ala Ala val 355 360 365 Gly Gly Arg Trp val Ser Gly Ala Ala Val Al a Arg His val Trp Asp 370 375 380 Ala Gln Ala Ser Gly Phe cys Gly Asp Leu Gly Arg Asp Glu Glu Pro 385 390 395 400 Ser Phe val Leu Leu Asp Thr Asp Ala Ala Gly Asp Gln Leu Phe Al a

    405 410 415

    2018203034 01 May 2018

    Thr Tyr Met Leu Asp 420 Pro Ala Arg Gly Ser Leu Leu Ser Ala Gly Thr 425 430 Pro Met His Phe Pro Arg Gly Gly Pro Ala Pro Gly Pro Asp Pro Ser 435 440 445 cys Trp Phe Asp Pro Asn Asn Ile cys Asp Gly Gly Leu Glu Pro Gly 450 455 460 Phe Ile Phe Leu Gly Phe Leu Leu val val Gly Met Gly Leu Ala Gly 465 470 475 480 Ala Leu Leu Ala His Tyr val Arg His Gln Leu Leu Hi s lie Gln Met 485 490 495 val Ser Gly Pro Asn Lys lie lie Leu Thr Val Asp Asp Ile Thr Phe 500 505 510 Leu Hi s Pro Hi s Gly Gly Ala Ser Arg Lys val Ala Gln Gly Ser Arg 515 520 525 Ser Ser Leu Ala Ala His ser Thr ser Asp lie Arg Ser Gly Pro ser 530 535 540 Gln Pro Ser Asp Ser pro Asn lie Gly Val Tyr Glu Gly Asp Arg val 545 550 555 560 Trp Leu Lys Lys Phe Pro Gly Glu Gln His Ile Ala Ile Arg Pro Al a 565 570 575 Thr Lys Thr Ala Phe Ser Lys Leu Gln Glu Leu Arg His Glu Asn val 580 585 590 Ala Leu Tyr Leu Gly Leu Phe Leu Ala Gln Gly Ala Glu Gly Pro Ala 595 600 605 Ala Leu Trp Glu Gly Asn Leu Ala val Val Ser Glu Hi s cys Thr Arg 610 615 620 Gly Ser Leu Gln Asp Leu Leu Ala Gln Arg Glu Ile Lys Leu Asp Trp 625 630 635 640 Met phe Lys ser ser Leu Leu Leu Asp Leu ile Lys Gly lie Arg Tyr 645 650 655 Leu His Hi s Arg Gly val Ala His Gly Arg Leu Lys Ser Arg Asn cys 660 665 670 lie val Asp Gly Arg Phe Val Leu Lys ile Thr Asp Hi s Gly Hi s Gly 675 680 685

    Arg Leu Leu Glu Ala Gln Lys val Leu Pro Glu Pro Pro Lys Ala Glu 32

    690

    695

    2018203034 01 May 2018

    Asp Gln 705

    Arg Arg

    Gln Glu

    Pro Asp

    Pro Phe 770

    Lys Gln 785

    Thr Phe

    Ile Asp

    Asp Leu

    Thr Asp 850

    Leu Lys 865

    Leu Tyr

    Glu Pro

    Asp Ala

    Asp Ala 930

    His Ala 945

    Gly Thr

    Leu Trp

    Gly Thr

    Val val 740

    Glu Val 755 val ser cys Trp

    Asp Leu

    Ser Met 820 lie Arg 835

    Arg Leu

    Thr Gly

    Phe Ser lie Glu 900 lie lie 915

    Tyr Met

    Ala Glu

    Phe Arg

    Thr

    Leu

    725 cys val

    Met

    Ala

    Phe

    805

    Leu

    Glu

    Leu

    Thr

    Asp

    885 val

    Gly val lie

    Met

    965

    Ala Pro 710

    Ala Gly

    Arg Ser

    Gln Arg

    Asp Gln 775

    Glu Gln 790

    Lys Asn

    Arg Met

    Arg Thr

    Thr Gln 855

    Pro val 870 lie val

    Val Asp

    Ser His

    Ala ser 935

    Ala Asn 950

    Arg His

    Glu Leu

    Leu Arg 715

    Asp val

    Phe Ser 730

    Ala Pro 745

    Tyr Ala

    700

    Asp Pro

    Leu Gly

    Met Leu val Arg 760

    Ala Pro

    Pro Glu

    Ile Asn

    Leu Glu 825

    Glu Glu 840

    Met Leu

    Glu Pro

    Gly Phe

    Leu Leu 905

    Asp val 920

    Gly Leu

    Met ser

    Met Pro

    Ser Pro val Glu

    Leu Arg 795

    Lys Gly 810

    Gln Tyr

    Leu Glu pro pro

    Glu Tyr 875

    Thr Thr 890

    Asn Asp

    Tyr Lys

    Pro Pro 765

    Cys lie 780

    Pro Ser

    Arg Lys

    Ser ser

    Leu Glu 845

    Ser val 860

    Phe Glu

    Ile Ser

    Leu Tyr val Glu 925

    Pro Gln

    Leu Asp 955

    Glu val 970

    Arg Asn 940 ile Leu

    Pro val

    Ala lie

    Glu

    750

    Leu

    Hi s

    Met

    Thr

    Asn

    830

    Lys

    Ala

    Gln

    Ala

    Thr

    910

    Thr

    Gly

    Ser

    Arg

    Leu Glu 720

    Ile Met 735

    Leu Thr

    Cys Arg

    Leu Met

    Asp Leu 800

    Asn lie 815

    Leu Glu

    Gln Lys

    Glu Ala val Thr 880

    Met Ser 895

    Leu Phe lie Gly

    Gln Arg

    Ala val 960

    Ile Arg 975

    2018203034 01 May 2018

    Ile Gly Leu His Ser Gly Pro Cys val Ala Gly Val Val Gly Leu Thr 980 985 990

    Met Pro Arg Tyr Cys Leu Phe Gly Asp Thr val Asn Thr Ala ser Arg 995 1000 1005

    Met Glu Ser Thr Gly Leu Pro Tyr Arg lie His Val Asn Leu Ser 1010 1015 1020

    Thr val Gly lie Leu Arg Ala Leu Asp Ser Gly Tyr Gln val Glu 1025 1030 1035

    Leu Arg Gly Arg Thr Glu Leu Lys Gly Lys Gly Ala Glu Asp Thr 1040 1045 1050

    Phe Trp Leu Val Gly Arg Arg Gly Phe Asn Lys Pro lie Pro Lys 1055 1060 1065 pro Pro Asp Leu Gln pro Gly Ala Ser Asn His Gly lie Ser Leu 1070 1075 1080

    Gln Glu Ile Pro Pro Glu Arg Arg Arg Lys Leu Glu Lys Ala Arg 1085 1090 1095

    Pro Gly Gln Phe Ser 1100 <210> 9 <211> 1012 <212> PRT <213> Ailuropoda melanoleuca <220>

    <221> misc_feature <222> (80)..(88) <223> Xaa can be any naturally occurring amino acid <220>

    <221> misc_feature <222> (964)..(971) <223> Xaa can be any naturally occurring amino acid <400> 9

    Met 1 Arg Ala cys Ala 5 Leu Leu Ala Gly Gly 10 Leu Pro Tyr Pro Arg 15 Leu cys Ala Pro Thr Arg Trp Ala Pro Ala Arg Pro Gly val Ser Arg Al a 20 25 30 Leu Pro Trp Pro Arg Pro Arg Leu Arg Leu Leu Leu Leu Leu Leu Leu 35 40 45 Arg Pro Pro ser val Leu Ser Ala Val Phe Thr val Gly val Leu Gly

    2018203034 01 May 2018

    50 55 60

    Pro Trp Ala Cys Asp Pro lie Phe Ala Arg Ala Arg Pro Asp Leu Xaa 65 70 75 80 xaa Xaa xaa Xaa xaa Xaa xaa xaa Asp Ala Leu Tyr Val Leu Leu Arg 85 90 95 Ala Phe Arg Trp Ala Arg val Ala Leu val Thr Ala Pro Gin Asp Leu 100 105 110 Trp val Glu Ala Gly Arg Ala Leu ser Ala Ala Leu Arg Ala Arg Gly 115 120 125 Leu Pro val Ala Leu val Thr Thr Met Glu Pro Ser Asp Leu Ser Gly 130 135 140 Al a Arg Glu Ala Leu Arg Arg val Gin Hi s Gly Pro Arg val Ser Ala 145 150 155 160 val ile Met val Met His ser val Leu Leu Gly Gly Glu Glu Gin Arg 165 170 175 cys Leu Leu Gin Ala Ala Glu Glu Leu Gly Leu Ala Asp Gly Ser Leu 180 185 190 val Phe Leu Pro Phe Asp Thr Leu Hi s Tyr Ala Leu Ser pro Gly Pro 195 200 205 Glu Ala Leu Ala Ala Leu Ala Asn ser Ser Gin Leu Arg Arg Ala Hi s 210 215 220 Asp Ala val Leu Thr Leu Thr Arg Hi s Cys pro Pro Gly Gly Ser val 225 230 235 240 Met Asp Ser Leu Arg Arg Ala Gin Glu Arg Gl n Glu Leu Pro Ser Asp 245 250 255 Leu Asn Leu Glu Gin val ser Pro Leu Phe Gly Thr lie Tyr Asp Al a 260 265 270 Val Phe Leu Leu Al a Gly Gly val Ala Arg Ala Arg Ala Al a Ala Al a 275 280 285 Asp ser Arg Val Pro Gly Phe cys Gly Ala Leu Gly Gly Ala Glu Gl u 290 295 300 Pro Pro Phe val Leu Leu Asp Thr Asp Ala Al a Gly Asp Arg Phe Phe 305 310 315 320 Ala Thr Tyr val Leu Asp Pro Thr Arg Gly Ser Leu Hi s Ser Ala Gly 325 330 335

    2018203034 01 May 2018

    Thr Pro val His 340 Phe Pro Arg Gly Gly Gly Ala Pro Gly 345 Pro 350 Asp Pro Ser Cys Trp Phe Glu Pro Asp Ser lie Cys Asn Gly Gly val Glu Pro 355 360 365 Gly Leu Val Phe Thr Gly Phe Leu Leu val val Gly Met Gly Leu Met 370 375 380 Gly Ala Phe Leu Ala Hi s Tyr val Arg Hi s Arg Leu Leu His Ile Gln 385 390 395 400 Met val ser Gly Pro Asn Lys lie lie Leu Thr Leu Asp Asp ile Thr 405 410 415 Phe Leu Hi s Pro Gln Gly Gly Ser Ala Arg Lys val Val Gln Gly Ser 420 425 430 Arg Ser Ser Leu Ala Ala Arg Ser Thr Ser Asp val Arg Ser Val Pro 435 440 445 Ser Gln Pro Ser Asp Gly Gly Asn ile Gly Leu Tyr Glu Gly Asp Trp 450 455 460 val Trp Leu Lys Lys Phe Pro Gly ser Gln Hi s Ile Ala ile Arg Pro 465 470 475 480 Ala Thr Lys Thr Ala Phe Ser Lys Leu Arg Glu Leu Arg His Glu Asn 485 490 495 val Ala Leu Tyr Leu Gly Leu Phe Leu Gly Gly Gly Glu Gly Gly Ser 500 505 510 Ala Ala Ala Gly Gly Gly Met Leu Ala Val val Ser Glu Hi s Cys Thr 515 520 525 Arg Gly Ser Leu Hi s Asp Leu Leu Ala Gln Arg Asp Ile Lys Leu Asp 530 535 540 Trp Met Phe Lys Ser Ser Leu Leu Leu Asp Leu lie Lys Gly Met Arg 545 550 555 560 Tyr Leu His His Arg Gly val Ala His Gly Arg Leu Lys Ser Arg Asn 565 570 575 cys val val Asp Gly Arg Phe val Leu Lys val Thr Asp Hi s Gly Hi s 580 585 590 Gly Arg Leu Leu Glu Al a Gln Lys val Leu Ala Glu Pro Pro Ser Al a

    595 600 605

    2018203034 01 May 2018

    Glu Glu 625 Asp Gln Leu 610 Arg Arg Gly Trp Thr Ala Pro Glu Leu 615 Leu Arg Asp Pro 620 Ala lie Leu ile 640 Thr Leu 630 Ala Gly Asp Val Phe 635 Ser Leu Gly Met Gln Glu Val Val cys Arg Ser Ser Pro Tyr Ala Met Leu Glu Leu 645 650 655 Ser Ala Arg Glu Val val Gln Arg Val Arg Ser Pro Pro Pro Leu Cys 660 665 670 Arg Pro ser val Ser Val Asp Gln Ala Pro Ala Glu cys Ile Gln Leu 675 680 685 Met Lys Gln cys Trp Ala Glu Gln Pro Glu Leu Arg Pro Ser Leu Asp 690 695 700 Arg Thr Phe Asp Gln Phe Lys Ser Ile Asn Lys Gly Arg Lys Thr Asn 705 710 715 720 Ile Ile Asp Ser Met Leu Arg Met Leu Glu Gln Tyr Ser Ser Asn Leu 725 730 735 Glu Gly Leu lie Arg Glu Arg Thr Glu Glu Leu Glu Leu Glu Lys Arg 740 745 750 Lys Thr Asp Arg Leu Arg Ala Ala Ser Leu Pro Ser Ser Val Ala Glu 755 760 765 Ala Leu Lys Met Gly Thr Pro val Glu Pro Glu Tyr Phe Glu Glu val 770 775 780 Thr Leu Tyr Phe Ser Asp lie val Gly Phe Thr Thr lie Ser Ala Met 785 790 795 800 Ser Glu Pro Ile Glu val val Asp Leu Leu Asn Asp Leu Tyr Thr Leu 805 810 815 Phe Asp Ala lie Ile Gly Ser His Asp val Tyr Lys val Glu Thr lie 820 825 830 Gly Asp Ala Tyr Met val Ala ser Gly Leu Pro Gln Arg Asn Gly Gln 835 840 845 Arg Hi s Ala Ala Glu Ile Ala Asn Met Ala Leu Asp Ile Leu Ser Ala 850 855 860 Val Gly Ser Phe Arg Met Arg Hi s Met Pro Glu Val Pro val Arg lie 865 870 875 880

    2018203034 01 May 2018

    Arg lie Gly Leu His Ser Gly Pro Cys Val 890 Ala Gly Val val Gly 895 Leu 885 Thr Met Pro Arg Tyr cys Leu Phe Gly Asp Thr val Asn Thr Ala Ser 900 905 910 Arg Met Glu Ser Thr Gly Leu Pro Tyr Arg Ile Hi s val Asn Met Ser 915 920 925 Thr val Arg ile Leu Arg Ala Leu Asp Glu Gly Phe Gln Thr Glu val 930 935 940 Arg Gly Arg Thr Glu Leu Lys Gly Lys Gly Ala Glu Asp Thr Tyr Trp 945 950 955 960 Leu val Gly xaa xaa Xaa Xaa xaa xaa Xaa xaa Pro ile Pro Lys Pro 965 970 975 Pro Asp Leu Gln Pro Gly Ala Ser Asn Hi s Gly lie ser Leu Gln Glu

    980 985 990 ile Pro Leu Asp Arg Arg Gln Lys Leu Glu Lys Ala Arg Pro Gly Gln 995 1000 1005

    Phe Ser Gly Lys 1010 <210> 10 <211> 1093 <212> PRT <213> Monodelphis domestica <400> 10

    Met 1 Leu Val Pro Ser 5 lie Asn Gly Leu Phe 10 His Hi s Pro Pro Trp 15 Cys Phe Pro Pro Leu Pro Leu Pro Leu Phe Phe Leu Phe Leu Leu Leu Leu 20 25 30 Leu pro val Pro val Leu Pro Ala Thr Phe Thr Ile Gly val Leu Gly 35 40 45 pro Trp Ser cys Asp Pro lie Phe Ser Arg Ala Arg Pro Asp Leu Al a 50 55 60 Ala Arg Leu Ala Ala Thr Arg Met Asn Hi s Asp Gln Ala Leu Glu Gly 65 70 75 80 Gly Pro Trp Phe Glu val Ile Leu Leu Pro Glu Pro cys Arg Thr Ser 85 90 95 Gly Ser Leu Gly Ala Leu Ser pro Ser Leu Ala Arg val ser Gly Leu

    100 105 110

    2018203034 01 May 2018

    Val Gly

    Gln Glu 130

    Ala Arg 145

    Ala Leu

    Pro Gln

    Arg Ser

    Asp Leu 210

    Lys Val 225

    Glu Glu

    Glu Gly

    Pro Pro

    Arg Lys 290

    Gly Ser 305

    Pro Leu

    Tyr Asp

    Ala Gly

    Pro Asn 370

    Pro

    115

    Ala

    Thr

    Leu

    Asp

    Arg

    195

    Glu

    Lys

    Gln

    Thr

    Gly

    275

    Ala val

    Asp

    Ala

    Gly

    355

    Thr

    Val Asn

    Gly Val

    Thr Ala

    Arg Ala 165

    Leu Trp 180

    Gly Leu

    Ser Ala

    Val Leu

    Arg Leu 245

    Met Val 260

    Pro Glu

    His Asp

    Ser Ala

    Leu Lys 325 ile Tyr 340

    Gly Gly

    Leu Val

    Pro

    Pro

    Pro

    150

    Phe

    Val

    Pro

    Lys

    Ile

    230

    Leu

    Phe

    Ala

    Ala ser

    310

    Pro

    Leu

    Trp

    Ser

    Ala Ala 120

    Leu val 135

    Ala Leu

    His Trp

    Glu Ala val Ala 200

    Asn Ala 215

    Met val

    Leu Glu

    Leu Pro

    Leu Arg 280 val Leu 295

    Leu Arg

    Gln Gln

    Leu Ala val ser 360

    Gly Phe 375

    Cys His

    Pro Trp

    Pro Leu

    Ala Lys 170

    Gly Gln 185

    Met val

    Leu Lys

    Met Hi s

    Ala Ala 250

    Phe Asp 265

    Pro lie

    Thr Leu

    Gln Ala

    Val Ser 330

    Gly Ala 345

    Gly Ala

    Cys Gly

    Pro

    Gly

    Ala

    155

    Val

    Ala

    Thr

    Arg

    Ser

    235

    Glu

    Thr

    Thr

    Thr

    Gln

    315

    Pro val

    Al a

    Asp

    Ala Glu 125 cys Pro 140

    Leu Asp

    Ala Leu

    Leu Ala

    Ser Leu 205

    Val Arg 220 val Leu

    Glu Leu

    Leu His

    Asn Ser 285

    Arg Tyr 300

    Glu His

    Leu Phe

    Ala Gly val Ala 365

    Leu Gly 380

    Leu Leu Ala

    Gln Gly Lys

    Ala Leu Tyr 160 ile Thr Ala 175

    Gly Gly Leu 190

    Glu Thr Thr

    Asp Gly pro

    Leu Gly Gly 240

    Gly Leu Val 255

    Tyr Ala Leu 270

    Ser Arg Leu

    Cys Pro Lys

    Arg Glu Leu 320

    Gly Thr lie 335

    Ala Gln val 350

    Arg His lie

    Gly Thr Lys

    2018203034 01 May 2018

    Glu 385 Pro Pro Phe val Leu Leu Asp 390 Thr Asp Gly Met Arg 395 Asp Gln Leu 400 Leu Pro Thr Tyr Thr Leu Asp Pro Ala Gln Gly val Leu His Hi s Ala 405 410 415 Gly Asn Pro lie Hi s Phe Pro His Gly Gly Gln Gly Pro Gly Pro Asp 420 425 430 Pro Pro Cys Trp Phe Asp Pro Asn val Ile cys Ser Gly Gly Ile Glu 435 440 445 Pro Arg Phe lie Leu Leu Val Ile Leu lie ile lie Gly Gly Gly Leu 450 455 460 Val Val Ala Thr Leu Ala Tyr Tyr val Arg Arg Gln Leu Leu His Ala 465 470 475 480 Gln Met val Ser Gly Pro Asn Lys Met lie Leu Thr Leu Glu Asp Ile 485 490 495 Thr Phe Phe Pro Arg Gln Gly Ser Ser Ser Arg Lys Ala Thr Glu Gly 500 505 510 Ser Arg Ser Ser Leu Ile Ala His Ser Ala Ser Asp Met Arg Ser lie 515 520 525 Pro Ser Gln Pro Pro Asp Asn Ser Asn lie Gly Met Tyr Glu Gly Asp 530 535 540 Trp Val Trp Leu Lys Lys Phe Pro Gly Glu His Tyr Thr Glu Ile Arg 545 550 555 560 Pro Ala Thr Lys Met Ala Phe Ser Lys Leu Arg Glu Leu Arg His Glu 565 570 575 Asn Val Ala val Gln Met Gly Leu Phe Leu Ala Gly Ser Met Glu Gly 580 585 590 Ala Ala Ala Gly Gly Leu Gly Gly Gly lie Leu Ala val val Ser Glu 595 600 605 Tyr cys Ser Arg Gly Ser Leu Gln Asp Leu Leu lie Gln Arg Asp lie 610 615 620 Lys Leu Asp Trp Met Phe Lys Ser Ser Leu Leu Leu Asp Leu ile Lys 625 630 635 640 Gly Leu Arg Tyr Leu Hi s Hi s Arg Gly val Al a Hi s Gly Arg Leu Lys 645 650 655

    2018203034 01 May 2018

    Ser Arg Asn Cys 660 Val Val Asp Gly Arg Phe 665 Val Leu Lys lie 670 Thr Asp Hi s Ala His Gly Arg Leu Leu Glu Ala Gln Arg Val Ser Leu Glu Pro 675 680 685 Pro Gln Ala Glu Asp Arg Leu Trp Thr Ala Pro Glu Leu Leu Arg Asn 690 695 700 Glu Ala Leu Glu Arg Gln Gly Thr Leu Gln Gly Asp Val Phe Ser val 705 710 715 720 Gly lie lie Met Gln Glu val val cys Arg Cys Glu Pro Tyr Ala Met 725 730 735 Leu Glu Leu Thr Pro Glu Glu lie lie Gln Lys val Gln Ser Pro pro 740 745 750 Pro Met cys Arg Pro Ser val Ser Val Asp Gln Ala Pro Met Glu cys 755 760 765 ile Gln Leu Met Lys Gln cys Trp Ala Glu Gln Pro Asp Leu Arg Pro 770 775 780 Asn Met Asp Thr Thr Phe Asp Leu Phe Lys Asn ile Asn Lys Gly Arg 785 790 795 800 Lys Thr Asn lie Ile Asp Ser Met Leu Arg Met Leu Glu Gln Tyr Ser 805 810 815 Ser Asn Leu Glu Asp Leu lie Arg Glu Arg Thr Glu Glu Leu Glu Leu 820 825 830 Glu Lys Gln Lys Thr Asp Lys Leu Leu Thr Gln Met Leu Pro Pro Ser 835 840 845 val Ala Glu Ala Leu Lys Leu Gly lie Pro val Glu Pro Glu Tyr Phe 850 855 860 Glu Glu Val Thr Leu Tyr Phe Ser Asp lie val Gly Phe Thr Thr lie 865 870 875 880 Ser Ala Met Ser Glu pro lie Glu val val Asp Leu Leu Asn Asp Leu 885 890 895 Tyr Thr Leu Phe Asp Al a lie lie Gly Ser Hi s Asp val Tyr Lys val 900 905 910 Glu Thr ile Gly Asp Ala Tyr Met val Ala ser Gly Leu Pro Lys Arg

    915 920 925

    Asn Gly Gln Arg His Ala Ala Glu Ile Ala Asn Met Ser Leu Asp Ile 41

    2018203034 01 May 2018

    930 935 940 Leu ser Ser Val Gly Ser Phe Arg Met Arg His Met Pro Glu val Pro 945 950 955 960 val Arg ile Arg lie Gly Leu His Ser Gly Pro Cys val Ala Gly val 965 970 975 val Gly Leu Thr Met Pro Arg Tyr cys Leu Phe Gly Asp Thr val Asn 980 985 990 Thr Ala Ser Arg Met Glu Ser Thr ι Sly Leu Pro Tyr Arg lie His val 995 1000 1005 Asn Leu Ser Thr val Lys Ile Leu Gln Gly Leu Asn Glu Gly Phe 1010 1015 1020 Gln ile Glu ile Arg Gly Arg Thr Glu Leu Lys Gly Lys Gly val 1025 1030 1035 Glu Asp Thr Tyr Trp Leu val Gly Arg Lys Gly Phe Asp Lys Pro 1040 1045 1050 Ile Pro ile Pro Pro Asp Leu Leu Pro Gly Ala Ser Asn His Gly 1055 1060 1065 lie Ser Leu Gln Glu ile Pro Glu Asp Arg Arg Lys Lys Leu Glu 1070 1075 1080 Lys Ala Arg Pro Gly Gln Pro Leu Gly Lys

    1085 1090 <210> 11 <211> 862 <212> PRT

    <213> Equus cabal 1 us <400> 11 Met val Met His Ser val Leu Leu Gly Gly Glu Glu Gln Arg cys Leu 1 5 10 15 Leu Glu Ala Ala Glu Glu Leu Gly Leu Ala Asp Gly Ser Leu val Phe 20 25 30 Leu Pro Phe Asp Thr Leu Hi s Tyr Ala Leu Ser Pro Gly Pro Glu Al a 35 40 45 Leu Ala Val Leu Al a Asn Asn ser Gln Leu Arg Arg Ala His Asp Al a 50 55 60 val Leu Thr Leu Thr Arg His cys Pro Leu Gly Gly Ser val Leu Asp 65 70 75 80

    2018203034 01 May 2018

    Ser Leu Arg Arg Ala 85 Gln Glu His Gln Glu Leu Pro ser Asp Leu Asn 90 95 Leu Gln Gln val Ser Pro Leu Phe Gly Thr Ile Tyr Asp Ala val Tyr 100 105 110 Leu Leu Ala Gly Gly Val Ala Arg Ala Arg Ala Ala Ala Gly Gly Ser 115 120 125 Trp Val Ser Gly Ala Ala val Ala His His val Arg Asp Ala Gln Val 130 135 140 Pro Gly Phe cys Gly Ala Leu Gly Gly Ala Glu Glu Pro Gln Phe Val 145 150 155 160 Leu Leu Asp Thr Asp Ala Ala Gly Asp Arg Leu Phe Ala Thr Tyr Met 165 170 175 Leu Asp Pro Thr Arg Gly Ser Leu Trp Ser Ala Gly Thr Pro Val Hi s 180 185 190 Phe Pro Arg Gly Gly Arg Gly Pro Gly Pro Asp Pro Trp cys Trp Phe 195 200 205 Asp Pro ASP Asp Ile cys Asn Gly Gly val Glu Pro Arg Leu Val Phe 210 215 220 lie Gly Phe Leu Leu Ala val Gly Met Gly Leu Ala Gly Val Phe Leu 225 230 235 240 Ala Hi s Tyr val Arg Hi s Arg Leu Leu Hi s Ile Gln Met Ala Ser Gly 245 250 255 Pro Asn Lys lie Ile Leu Thr Leu Asp Asp Ile Thr Phe Leu His Pro 260 265 270 Gln Gly Gly Ser Ser Arg Lys val lie Gln Gly Ser Arg Ser Ser Leu 275 280 285 Al a Ala Arg Ser Val Ser Asp Ile Arg Ser val Pro Ser Gln Pro Met 290 295 300 Asp Ser Ser Asn lie Gly Leu Tyr Glu Gly Asp Trp val Trp Leu Lys 305 310 315 320 Lys Phe Pro Gly Asp Gln Hi s lie Ala lie Arg Pro Ala Thr Lys Thr 325 330 335 Ala Phe Ser Lys Leu Arg Glu Leu Arg His Glu Asn val Ala Leu Tyr 340 345 350

    2018203034 01 May 2018

    Leu Gly Leu 355 Phe Leu Ala Gly Gly Ser ser 360 Gly Ala Ala 365 Al a Pro Arg Glu Gly Met Leu Ala Val val Ser Glu His Cys Ala Arg Gly Ser Leu 370 375 380 Hi s Asp Leu Leu Ala Gin Arg Asp Ile Lys Leu Asp Trp Met Phe Lys 385 390 395 400 Ser Ser Leu Leu Leu Asp Leu Ile Lys Gly Met Arg Tyr Leu Hi s Hi s 405 410 415 Arg Gly val Ala His Gly Arg Leu Lys Ser Arg Asn Cys val val Asp 420 425 430 Gly Arg Phe val Leu Lys val Thr Asp Hi s Gly His Gly Arg Leu Leu 435 440 445 Glu Ala Gin Lys val Leu Pro Glu Pro Pro Ser Ala Glu Asp Gin Leu 450 455 460 Trp Thr Ala Pro Glu Leu Leu Arg Asp pro Al a Leu Glu Arg Gin Gly 465 470 475 480 Thr Leu Ala Gly Asp Val Phe Ser Leu Gly lie lie lie Gin Glu val 485 490 495 Val Cys Arg Ser Thr Pro Tyr Ala Met Leu Glu Leu Thr Pro Glu Glu 500 505 510 val Val Gin Arg Leu Gin Ser Pro Pro Pro Leu cys Arg Pro Ser Val 515 520 525 Ser Met Asp Gin Ala Pro Met Glu cys lie Gin Leu Met Lys Gin cys 530 535 540 Trp Ala Glu Gin Pro Asp Leu Arg pro ser Met ASp Arg Thr Phe ASp 545 550 555 560 Leu Phe Lys Ser ile Asn Lys Gly Arg Lys Thr Asn ile Ile Asp Ser 565 570 575 Met Leu Arg Met Leu Glu Gin Tyr ser Ser Asn Leu Glu Asp Leu lie 580 585 590 Arg Glu Arg Thr Glu Glu Leu Glu Leu Glu Lys Gin Lys Thr Asp Arg 595 600 605 Leu Leu Thr Gin Met Leu Pro Pro Ser Val Ala Glu Ala Leu Lys Met 610 615 620 Gly Thr Pro Val Glu Pro Glu Tyr Phe Glu Glu val Thr Leu Tyr Phe

    2018203034 01 May 2018

    625 630 635 640 Ser Asp lie val Gly Phe Thr Thr Ile Ser Ala Met Ser Glu Pro lie 645 650 655 Glu val Val Asp Leu Leu Asn Asp Leu Tyr Thr Leu Phe Asp Ala lie 660 665 670 Ile Gly Ser Hi s Asp Val Tyr Lys val Glu Thr lie Gly Asp Ala Tyr 675 680 685 Met val Ala Ser Gly Leu Pro Gln Arg Asn Gly Gln Arg His Ala Ala 690 695 700 Glu Ile Ala Asn Met Ala Leu Asp Ile Leu Ser Ala val Gly Ser Phe 705 710 715 720 Arg Met Arg Hi s Met Pro Glu val Pro val Arg lie Arg ile Gly Leu 725 730 735 His Ser Gly Pro Cys val Ala Gly Val Val Gly Leu Thr Met Pro Arg 740 745 750 Tyr Cys Leu Phe Gly Asp Thr val Asn Thr Ala Ser Arg Met Glu Ser 755 760 765 Thr Gly Leu Pro Tyr Arg Ile Hi s val Asn Met Ser Thr Val Arg lie 770 775 780 Leu Arg Ala Leu Asp Glu Gly Phe Gln Val Glu Val Arg Gly Arg Thr 785 790 795 800 Glu Leu Lys Gly Lys Gly val Glu Asp Thr Tyr Trp Leu Val Gly Arg 805 810 815 Arg Gly Phe Asn Lys Pro Ile Pro Lys Pro Pro Asp Leu Gln Pro Gly 820 825 830 Ala Ser Asn His Gly Ile ser Leu Gln Glu lie Pro Pro Glu Arg Arg 835 840 845 Gln Lys Leu Glu Lys Al a Arg Pro Gly Gln Phe Ser Gly Lys 850 855 860

    <210> 12 <211> 292 <212> DNA <213> Homo sapiens <400> 12 gggccccaga agcctggtgg ttgtttgtcc ttctcagggg aaaagtgagg cggccccttg gaggaagggg ccgggcagaa tgatctaatc ggattccaag cagctcaggg gattgtcttt

    120

    2018203034 01 May 2018

    ttctagcacc ttcttgccac tcctaagcgt cctccgtgac cccggctggg atttagcctg 180 gtgctgtgtc agccccggtc tcccaggggc ttcccagtgg tccccaggaa ccctcgacag 240 ggcccggtct ctctcgtcca gcaagggcag ggacgggcca caggccaagg gc 292 <210> 13 <211> 953 <212> DNA <213> Artificial Sequence <220> <223> hybrid cba/cmv promoter <400> 13 aattcggtac cctagttatt aatagtaatc aattacgggg tcattagttc atagcccata 60 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 120 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt 180 ccattgacgt caatgggtgg actatttacg gtaaactgcc cacttggcag tacatcaagt 240 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca 300 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt 360 catcgctatt accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc 420 cccctcccca cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg 480 ggcggggggg gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg 540 ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt 600 tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt 660 cgctgcgacg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc 720 ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg 780 gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc 840 ttgaggggct ccgggagcta gagcctctgc taaccatgtt catgccttct tctttttcct 900 acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca aag 953

    <210> 14 <211> 36 <212> DNA <213> Artificial Sequence <22O> <223> Forward Primer <400> 14 aaaagcggcc gcatgagcgc ttggctcctg ccagcc 36 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer

    2018203034 01 May 2018 <400> 15 aaaagcggcc gctcacttcc cagtaaactg gcctgg 36 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <220>

    <223> Forward Primer <400> 16 gacccttcct gctggttcga tcca 24 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220>

    <223> Reverse Primer <400> 17 ctgcatgtgt agcagcctgt gcctc 25 <210> 18 <211> 992 <212> PRT <213> Artificial Sequence <220>

    <223> Consensus Sequence

    <400> : 18 Met Ser Ala Ala Gly Gly Leu Gly cys Pro Arg Ala Pro ser lie Pro 1 5 10 15 Arg Leu Leu Leu Leu Leu Leu Leu Ser Leu Ser Ala Val Phe Val Gly 20 25 30 Val Leu Gly Pro Trp Ala cys Asp Pro lie Phe Al a Arg Ala Arg Pro 35 40 45 Asp lie Ala Ala Arg Leu Ala Ala Arg Leu Asn Ala Leu Asp Gly Gly 50 55 60 Pro Arg Phe Glu val Ala Leu Leu Pro Glu pro Cys Thr pro Gly Ser 65 70 75 80 Leu Gly Ala Val ser Ser Ala Ser Arg val Ser Gly Leu val Gly Pro 85 90 95 val Asn Pro Ala Ala cys Arg pro Ala Glu Leu Leu Ala Gln Glu Al a 100 105 110 Gly val Ala Leu val Pro Trp Gly val Pro Gly Thr Arg Ala Ala Gly 115 120 125

    2018203034 01 May 2018

    Thr Thr 130 Ala Pro Val Thr Pro 135 Ala Ala Asp Ala Leu 140 Tyr Leu Leu Arg Ala Phe Arg Trp Ala Val Ala Leu Ile Thr Ala Pro Gln Asp Leu Trp 145 150 155 160 Val Glu Ala Gly Ala Leu Ser Thr Ala Leu Arg Ala Arg Gly Leu Pro 165 170 175 Val Ala Leu Val Thr ser Met Glu Val Arg val Ile Met Val Met Gly 180 185 190 ser val Leu Leu Gly Gly Glu Glu Gln Arg Leu Leu Glu Ala Ala Gl u 195 200 205 Glu Leu Ala Leu Asp Gly Ser Leu Val Phe Leu Pro Phe Asp Thr Leu 210 215 220 Hl s Trp Ala Leu ser Pro Gly Pro Asp Ala lie Ala Asn Ser Ser Gln 225 230 235 240 Leu Arg Lys Ala Hi s Asp Ala Val Leu Thr Leu Thr Arg Cys Pro Gly 245 250 255 Gly Ser Val Asp Ser Leu Arg Arg Ala Gln Glu His Glu Leu Pro Leu 260 265 270 Asp Leu Asn Leu Gln val Ser Pro Leu Phe Gly Thr ile Tyr Asp Ala 275 280 285 Val Phe Leu Leu Ala Gly Gly Thr Ala Thr Al a Gly Gly Gly Trp Val 290 295 300 ser Gly Ala Ala val Ala Arg Ile Arg Asp Al a Val Gly Phe Cys Gly 305 310 315 320 Leu Gly Glu Glu Pro Ser Phe Val Leu Ile Asp Thr Asp Ala Ser Gly 325 330 335 Asp Gln Leu Phe Ala Thr Hi s Leu Leu Asp Pro Gly Ser Ala Gly Thr 340 345 350 pro Met Hi s Phe pro Lys Gly Gly Ala Pro Gly Pro Asp Pro Ser Cys 355 360 365 Trp Phe Asp Pro Asp lie cys Asn Gly Gly Val Glu Pro Leu val Phe 370 375 380 lie Gly Phe Leu Leu val Ile Gly Met Gly Leu Gly Ala Phe Leu Ala 385 390 395 400

    2018203034 01 May 2018

    Phe Leu Ala His Tyr 405 Arg Hi s Arg Leu Leu 410 Hi s Ile Gln Met Ser 415 Gly Pro Asn Lys Ile Ile Leu Thr Leu Asp Asp Ile Thr Phe Leu Hi s Pro 420 425 430 Gly Gly Ser Arg Lys Val Gln Gly Ser Arg Ser Ser Leu Ala Arg Ser 435 440 445 Ser Asp Ile Arg Ser Ile Ser Gln Asp Thr Asn lie Gly Leu Tyr Glu 450 455 460 Gly Asp Trp Val Trp Leu Lys Lys Phe Pro Gly Asp His ile Ala ile 465 470 475 480 Arg Pro Ala Thr Lys Ala Phe Ser Lys lie Arg Glu Leu Arg Hi s Glu 485 490 495 Asn val Ala Leu Tyr Leu Gly Leu Phe Leu Ala Gly Ala Gly Ala Pro 500 505 510 Ala Pro Gly Glu Gly Ile Leu Ala val val Ser Glu His Cys Ala Arg 515 520 525 Gly ser Leu Asp Leu Leu Ala Gln Arg Asp lie Lys Leu Asp Trp Met 530 535 540 Phe Lys Ser Ser Leu Leu Leu Asp Leu lie Lys Gly Ile Arg Tyr Leu 545 550 555 560 Hi s Hi s Arg Gly Val Al a Hi s Gly Arg Leu Lys Ser Arg Asn cys Val 565 570 575 val Asp Gly Arg Phe val Leu Lys val Thr ASp Hi s Gly Hi s Gly Arg 580 585 590 Leu Leu Glu Ala Gln Arg val Leu Pro Glu Pro Pro ser Ala Glu Asp 595 600 605 Gln Leu Trp Thr Ala Pro Glu Leu Leu Arg Asp Pro Leu Glu Arg Arg 610 615 620 Arg Gly Thr Leu Ala Gly ASP val phe Ser Leu Ala lie lie Met Gln 625 630 635 640 Glu Val val cys Arg Ser Pro Tyr Ala Met Leu Glu Leu Thr Pro Glu 645 650 655 Glu Val ile Arg val Ser Pro Pro pro Leu cys Arg Pro val Ser Ile

    660 665 670

    Asp Gln Ala Pro Met Glu Cys Ile Gln Leu Met Gln val Trp Ala Glu 49

    2018203034 01 May 2018

    675 680 685

    Pro Glu Leu Arg Pro ser Met Asp Thr Phe Asp Leu Phe Lys Ser Ile 690 695 700 Asn Lys Gly Arg Lys Asn Ile lie Asp Ser Met Leu Arg Met Leu Glu 705 710 715 720 Gln Tyr Ser Ser Asn Leu Glu Asp Leu ile Arg Glu Arg Thr Glu Glu 725 730 735 Leu Glu Glu Glu Lys Gln Lys Thr Asp Arg Leu Leu Thr Gln Met Leu 740 745 750 Pro Pro Ser val Ala Glu Ala Leu Lys Met Gly Thr val Glu Pro Glu 755 760 765 Tyr Phe Glu Glu val Thr Leu Tyr Phe Ser Asp lie val Gly Phe Thr 770 775 780 Thr ile Ser Ala Met Ser Glu Pro lie Glu val val Asp Leu Leu Asn 785 790 795 800 Asp Leu Tyr Thr Leu Phe Asp Ala Ile Ile Gly Ala Hi s Asp val Tyr 805 810 815 Lys val Glu Thr Ile Gly Asp Ala Tyr Met val Ala Ser Gly Leu Pro 820 825 830 Gln Arg Asn Gly Arg Hi s Ala Ala Glu lie Ala Asn Met Ala Leu Asp 835 840 845 lie Leu Ser Ala val Gly Ser Phe Arg Phe Arg Met Arg His Met Pro 850 855 860 Glu val Pro Val Arg lie Arg lie Gly Leu Hi s Ser Gly Pro cys val 865 870 875 880 Ala Gly val val Gly Leu Thr Met Pro Arg Tyr cys Leu Phe Gly Asp 885 890 895 Thr val Asn Thr Ala Ser Met Glu ser Thr Gly Leu Pro Tyr Arg Ile 900 905 910 His val Asn Ser Thr val lie Leu Ala Leu Gly Phe Glu Arg Gly Arg 915 920 925 Thr Glu Leu Lys Gly Lys Gly Glu Asp Thr Tyr Trp Leu Val Gly Arg 930 935 940 Gly Phe Asn Lys Pro lie Pro Lys Pro Pro Asp Leu Gln Pro Gly Ala 945 950 955 960

    2018203034 01 May 2018

    Ser Asn His Gly He Ser Leu His Gly lie ser Leu Glu lie pro Pro 965 970 975

    Asp Arg Arg Lys Leu Glu Lys Ala Arg Pro Gly Gln Phe Ser Gly Lvs 980 985 990