COMPOSITIONS AND METHODS FOR TREATMENT OF DOMINANT

RETINITIS PIGMENTOSA

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing dates of U.S. Provisional Application No. 62/679,585, filed June 1, 2018, and U.S. Provisional Application No. 62/809,539, filed February 22, 2019, the entire contents of each of which are incorporated by reference.

BACKGROUND

Autosomal dominant retinitis pigmentosa (adRP) is a blinding disease affecting 1 in 12,000 people. A sizeable fraction of these individuals carry a mutation in the rhodopsin gene (RHO), the light harvesting pigment protein of the photoreceptor cells in the retina. The disease is dominant because inheritance of the mutated gene from either parent leads to retinal degeneration and eventual blindness. Over 100 different mutations identified in RHO lead to blindness. There is currently no approved drug or gene therapy treatment for adRP. Thus, there is a need for effective treatment options pertaining to any and all causes of adRP and related conditions.

SUMMARY

Aspects of the disclosure relate to compositions and methods for treating retinitis pigmentosa (e.g., dominant retinitis pigmentosa) in a subject (e.g., in a human). In some embodiments, one or both alleles of the rhodopsin gene ( RHO gene) of a subject (e.g., a human) are silenced by administering a short hairpin RNA (shRNA) molecule to a subject (e.g., to a subject having retinitis pigmentosa, for example to a human having dominant retinitis pigmentosa). In some embodiments, a replacement RHO coding sequence also is administered to the subject to provide a functional RHO protein, e.g., to restore photoreceptor function to the subject. In some embodiments, the replacement RHO coding sequence has one or more nucleotide substitutions relative to the endogenous gene allele(s) that render the replacement gene resistant to the effects of the interfering RNA. In some embodiments, the replacement RHO coding sequence is a human RHO coding sequence (e.g., a wild-type human RHO coding sequence) that includes one or more (e.g., 1, 2, 3, 4, 5, or more) substitutions to render the gene resistant (also referred to as“hardened”) to degradation mediated by the shRNA. In some aspects, the disclosure provides a short hairpin RNA (shRNA) comprising a sense strand comprising the nucleotide sequence GUGGCAUUCUACAUCUUCA (SEQ ID NO: 1), an antisense strand comprising the nucleotide sequence

UGAAGAUGUAGAAUGCCAC (SEQ ID NO: 2), and a loop comprising the nucleotide sequence UUCAAGAGA (SEQ ID NO: 3).

In some aspects, the disclosure provides a short hairpin RNA (shRNA) comprising a sense strand comprising the nucleotide sequence GErGGCAUErCETACAUCETErCA (SEQ ID NO: 1), an antisense strand comprising the nucleotide sequence

UGAAGAUGUAGAAUGCCAC (SEQ ID NO: 2), and a loop. The loop may consist of nine nucleotides.

In some embodiments, the shRNA comprises the nucleotide sequence:

GU GGC AUUCU AC AUCUU C AUUC A AG AG AU G A AG AU GU AG A AU GCC AC (SEQ ID NO: 4). In some embodiments, the shRNA consists of the nucleotide sequence

GU GGC AUUCU AC AUCUU C AUUC A AG AG AU G A AG AU GU AG A AU GCC AC (SEQ ID NO: 4). In some embodiments, the shRNA comprises the nucleotide sequence:

GUGGCAUUCUACAUCUUCAUUCAAGAGAUGAAGAUGUAGAAUGCCACUU (SEQ ID NO: 36). In some embodiments, the shRNA consists of the nucleotide sequence

GUGGCAUUCUACAUCUUCAUUCAAGAGAUGAAGAUGUAGAAUGCCACUU (SEQ ID NO: 36).

In some aspects, the disclosure provides a short hairpin RNA (shRNA) molecule comprising: a) a sense and antisense strand comprising one of the following sets of sequences: i) a sense strand comprising the nucleotide sequence

CUGCCUACAUGUUUCUGCU (SEQ ID NO: 21) and an antisense strand comprising the nucleotide sequence AGCAGAAACAUGUAGGCAG (SEQ ID NO: 22); ii) a sense strand comprising the nucleotide sequence CCUACAUGUUUCUGCUGAU (SEQ ID NO: 23) and an antisense strand comprising the nucleotide sequence AUCAGCAGAAACAUGUAGG (SEQ ID NO: 24); iii) a sense strand comprising the nucleotide sequence

GCAUGGUCAUCAUCAUGGU (SEQ ID NO: 25) and an antisense strand comprising the nucleotide sequence ACC AU GAU GAU GACC AU GC (SEQ ID NO: 26); or iv) a sense strand comprising the nucleotide sequence GUGGCAUUCUACAUCUUCA (SEQ ID NO: 1) and an antisense strand comprising the nucleotide sequence UGAAGAUGUAGAAUGCCAC (SEQ ID NO: 2); and b) a loop comprising the nucleotide sequence UUCAAGAGA (SEQ ID NO: 3). In some aspects, the disclosure provides a short hairpin RNA (shRNA) molecule comprising: a) a sense and antisense strand comprising one of the following sets of sequences: i) a sense strand comprising the nucleotide sequence

CUGCCUACAUGUUUCUGCU (SEQ ID NO: 21) and an antisense strand comprising the nucleotide sequence AGCAGAAACAUGUAGGCAG (SEQ ID NO: 22); ii) a sense strand comprising the nucleotide sequence CCUACAUGUUUCUGCUGAU (SEQ ID NO: 23) and an antisense strand comprising the nucleotide sequence AUCAGCAGAAACAUGETAGG (SEQ ID NO: 24); iii) a sense strand comprising the nucleotide sequence

GCAUGGETCAUCAUCAUGGET (SEQ ID NO: 25) and an antisense strand comprising the nucleotide sequence ACCAUGAUGAUGACCAUGC (SEQ ID NO: 26); or iv) a sense strand comprising the nucleotide sequence GErGGCAUErCErACAUCErErCA (SEQ ID NO: 1) and an antisense strand comprising the nucleotide sequence UGAAGAUGErAGAAUGCCAC (SEQ ID NO: 2); and b) a loop consisting of nine nucleotides.

In some embodiments, the short hairpin RNA (shRNA) molecule comprising or consists of one of the following nucleotide sequences:

CUGCCUACAUGUUUCUGCUUUCAAGAGAAGCAGAAACAUGUAGGCAG (SEQ ID NO: 27); CCUACAUGUUUCUGCUGAUUUCAAGAGAAUCAGCAGAAACAUGUAGG (SEQ ID NO: 28);

GC AU GGUC AU C AUC AU GGUUU C A AG AG A ACC AU G AU G AU G ACC AU GC (SEQ ID NO: 29); or

GU GGC AUUCU AC AUCUU C AUUC A AG AG AU G A AG AU GU AG A AU GCC AC (SEQ ID NO: 4). In some embodiments, the short hairpin acid (shRNA) molecule comprising or consists of one of the following nucleotide sequences:

CUGCCUACAUGUUUCUGCUUUCAAGAGAAGCAGAAACAUGUAGGCAGUU (SEQ ID NO: 37);

CCUACAUGUUUCUGCUGAUUUCAAGAGAAUCAGCAGAAACAUGUAGGUU (SEQ ID NO: 38);

GC AU GGUC AUC AUC AU GGUUUC AAGAG AACC AU GAU GAU GACC AU GCUU (SEQ ID NO: 39); or

GUGGCAUUCUACAUCUUCAUUCAAGAGAUGAAGAUGUAGAAUGCCACUU (SEQ ID NO: 36).

In other aspects, the disclosure provides a vector encoding an shRNA of any one of the above-mentioned embodiments or as otherwise described herein. In some embodiments, the shRNA coding sequence is operably linked to a promoter, e.g., a human Hl RNA promoter.

In some embodiments, the vector further comprises a recombinant RHO coding sequence that does not contain a sequence targeted by the shRNA. In some embodiments, the recombinant RHO coding sequence is codon-optimized for expression in a human cell. In some embodiments, the recombinant RHO coding sequence comprises a nucleotide sequence that is at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the recombinant RHO coding sequence comprises a nucleotide sequence that is one, two, three, four, five or between five and ten nucleotides different from the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the recombinant RHO coding sequence comprises the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the recombinant RHO coding sequence is operably linked to a promoter, e.g., a human opsin proximal promoter. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a viral vector. In some

embodiments, the viral vector is a recombinant adeno-associated viral (rAAV) vector. In some embodiments, the rAAV vector is self-complementary.

In other aspects, the disclosure provides a vector comprising a nucleotide sequence that is at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical to the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the recombinant RHO coding sequence comprises a nucleotide sequence that is one, two, three, four, five or between five and ten nucleotides different from the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the vector comprises the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a recombinant adeno-associated viral (rAAV) vector.

In some embodiments, the rAAV vector is self-complementary.

In yet other aspects, the disclosure provides a recombinant adeno-associated viral (rAAV) particle comprising any one of the rAAV vectors described above or as otherwise described herein. In some embodiments, the rAAV particle is an rAAV serotype 5 (rAAV5) particle.

In other aspects, the disclosure provides a composition comprising any one of the shRNAs, vectors, or rAAV particles described above or as otherwise described herein and a pharmaceutically acceptable carrier. In some embodiments, the disclosure provides a method of modulating RHO expression in a subject (e.g., a human subject), the method comprising administering to the subject the composition. In some embodiments, the disclosure provides a method of treating retinitis pigmentosa in a subject (e.g., a human subject), the method comprising administering to the subject the composition. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a rodent or a dog. In some

embodiments, the mammal is a human (e.g., a human having or known to have, for example diagnosed as having, retinitis pigmentosa, for example dominant retinitis pigmentosa). In some embodiments, the composition is for use in treating retinitis pigmentosa. In some embodiments, the composition is for use in the manufacture of a medicament to treat retinitis pigmentosa.

These and other aspects are described in the following drawings, examples, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. It is to be understood that the data illustrated in the drawings in no way limit the scope of the disclosure.

FIGs. 1A-1H show shRNA-mediated knockdown of wild type (WT), P23H, T17M, and shRNA820-resistant ( RHOsio ) variants of human rhodopsin ( RHO ). HEK293T cells were transfected with a plasmid expressing either WT, P23H, T17M or shRNA820-resistant ( RHO820 ) human RHO with a C-terminal turboGFP tag ( RHO-tGFP ) as well as with a rAAV2 plasmid (denoted with lane labels) encoding either empty DNA (no shRNA), a control shRNA, shRNA , shRNA i _. , or shRNAsio . A no DNA transfection control was also included. (FIGs. 1A, 1C, 1E, 1G) Immunoblots of protein samples isolated from transfected HEK293T cells probed for turboGFP tag and b-tubulin as the loading control. (FIGs. 1B, 1D, 1F, 1H) Relative quantification of the monomeric form of RHO-GFP (top row) and of the monomeric and aggregated forms of RHO-GFP (lower row). The first lane of each western blot contained the Chameleon Duo Pre- stained Protein Fadder from Fi-Cor. Bars denote mean value of three technical replicates, error bars denote SEM. ns. = not significant, *p<0.05, **p<0.0l, ***p<0.00l, ****p<0.000l. FIGs. 2A-2H show suppression of rhodopsin with shRNAsx ₎ in wildtype (WT) retinas. (FIGs. 2A-2C) In vivo imaging results from representative WT eyes 7-8 weeks post injection with scAAV2/5-H 1 -shRNAnin at lxlO ¹¹ (FIG. 2B) and 5xl0 ⁿ vg/mL (FIG. 2C) titers as compared to uninjected control (FIG. 2A). Shown are OCT scans (left), normalized inner/outer segment (IS/OS) intensity topography (middle) and outer nuclear layer (ONL) thickness topography (right). Dotted lines, injection bleb boundaries. Arrows, location of the OCTs shown on left. (FIGs. 2D, 2E) Normalized IS/OS intensity (FIG. 2D) and ONL thickness (FIG. 2E) sampled within the injected blebs (horizontal line shaded circles, upper panels) and uninjected control locations (diagonal line shaded circles, lower panels) in ten eyes injected with a range of titers. Symbols represent group averages (± SD) from 33 to 95 samples (see FIG. 12). Dashed lines denote the 99 ^th percentile limits of the respective parameters sampled at the same retinal locations in uninjected control eyes. Dropdown arrows estimate the titers corresponding to the transitions to a detectable effect. (FIG. 2F) Microphotographs of hematoxylin/eosin stained (top row) and rhodopsin (RHO)

immunolabeled retinal cryo sections showing morphology of the ONL and outer segments (OS) in treated (Tx; with l-lOxlO ¹¹ vg/mL titer range) and untreated (UnTx) areas 7-8 weeks post- injection. (FIG. 2G) Schematic representation of the retinas of WT dogs treated with 1- 50 xlO ¹¹ vg/mL titers showing the location of neuroretinal punches used for quantification of rhodopsin (RNA and protein) expression 7-8 weeks post-injection. Dashed lines, bleb boundaries; stippled area: tapetal region. (FIG. 2H) Quantification of the levels of endogenous canine RHO RNA remaining in the treated retinal area as a percentage of levels measured in the untreated area of eyes injected with the different vector titers. Representative immunoblot and quantification of the levels of endogenous canine RHO protein remaining in the treated retinal area as a percentage of levels measured in the untreated area of eyes injected with the different vector titers. Labels such as N282-OD refer to animal and eye; OSasOD designation refers to left eye being displayed as right eye for comparability.

FIGs. 3A-3D show suppression of rhodopsin with shRNAsio in ////G- mutant retinas. (FIG. 3A) Schematic representation of the fundus of four RHO-mutant dog eyes injected with scAAV2/5-H 1 -shRNAnx ₎ at 1-10 xlO ¹¹ vg/mL titers showing the location of neuroretinal punches used for quantification of rhodopsin (RNA and protein) expression at 8-10 weeks post-injection. Dashed lines, bleb boundaries; stippled area: tapetal region. (FIG. 3B) Quantification of the levels of endogenous canine RHO RNA remaining in the treated retinal area as a percentage of levels measured in the untreated area of eyes injected with different titers. Representative immunoblot and quantification of the levels of endogenous canine RHO protein remaining in the treated retinal area as a percentage of levels measured in the untreated area of eyes injected with 1- 50 xlO ¹¹ vg/mL titers. (FIG. 3C) Outer nuclear layer (ONL) thickness topography two weeks post light exposure (8-10 weeks post- injection) in four RHO- mutant dog eyes treated with 1-10 xlO ¹¹ vg/mL titers. Dotted lines, bleb boundaries; dashed lines, ONL rescue boundaries. Insets, maps of significance showing retinal regions with ONL thickness (left) and IS/OS intensity (right) values compared point by point to the 99 ^th percentile confidence intervals of uninjected controls. (FIG. 3D)

Microphotographs of H&E stained (top row) and rhodopsin (RHO,)/human cone arrestin (hCA) co-immunolabeled retinal cryosections showing morphology of the ONL and outer segments (OS) two weeks post light exposure (8-10 weeks post-injection) in treated (Tx; with l-lOxlO ¹¹ vg/mL titer range) and untreated (UnTx) areas of the same eyes shown in (FIG. 3C).

FIGs. 4A-4G show suppression and replacement of rhodopsin with single vector prevents retinal degeneration in //7/G-mutant retinas. (FIGs. 4A-4B) Outer nuclear layer (ONL) thickness topography after injection/before light exposure (post Inj.) and two weeks post light exposure (post LE) in two //7/G-mutant eyes injected with scAAV2/5-hOP- RHO _S20 -H1- shRNAsio at 5 xlO ¹¹ vg/mL titer. Dotted lines, bleb boundaries; dashed lines, ONL rescue boundaries. Insets, maps of significance described in FIGs. 3A-3E. (FIGs. 4C- 4D) Rhodopsin (RHO)/human cone arrestin (hCA) co-immunolabeled retinal cryosections showing morphology of the ONL and outer segments (OS) in treated and untreated (UnTx) areas of the same eyes shown in (FIGs. 4A-4B). (FIG. 4E) Schematic representation of the fundus of four //7/G-mutant dog eyes injected with a 5xl0 ⁿ vg/mL titer showing the location of neuroretinal punches used for quantification of rhodopsin (RNA and protein) expression. Dashed lines, bleb boundaries; stippled area: tapetal region. (FIG. 4F) Quantification of the levels of endogenous canine RHO RNA remaining in the treated retinal area as a percentage of levels measured in the untreated area of injected eyes. (FIG. 4G) Quantification of the levels of exogenous human RHO RNA ( RHOsio ) present in the treated retinal area as a percentage of physiological levels of endogenous canine RHO measured in the untreated area of injected eyes. Representative immunoblot and quantification of the levels of total

(endogenous canine and RHOsio ) RHO protein remaining in the treated retinal area as a percentage of levels measured in the untreated area of injected eyes.

FIGs. 5A-5D show protection of retinal structure and function in RHO-mutant retinas treated with single vector that combines suppression and replacement of rhodopsin of up to 5 weeks. (FIG. 5A) Upper, Timeline showing time-points of injection of scANV2/5-RHOs ₂₀ - shRNAsio (5 xlO ¹¹ vg/mL titer) in one eye of two /7/70-mutant dogs (contralateral eye injected with BSS), light exposures (LE1-LE4), OCT imaging and ERG sessions. Lower , Representative outer nuclear layer (ONL) thickness maps prior to injection, 11 weeks post injection (immediately before LE1), 1.5 weeks post LE1, and 2.1 weeks post LE4 of an eye injected with scAAY2/5-RHOs ₂₀ -shRNAs ₂₀ . Dashed lines, ONL rescue boundaries. The optic nerve head (black circle) and major blood vessels (solid black line), tapetum boundary (solid grey line), and fovea-like region (ellipse) are overlaid (middle row). Insets, maps of significance described in FIGs. 3A-3E. (FIG. 5B) Schematics (left) showing retinal locations sampled for quantification of ONL thickness and IS/OS intensity within the treated area of two RHO mutant eyes injected with sc A AY 215- RHOS _K) -shRNAsio . Longitudinal

quantification of the mean (± SD) difference in ONL thickness (middle) and IS/OS intensity (right) in the injected eyes when compared to uninjected controls. Horizontal dashed lines represent limits of WT variability (+/-3SD). (FIG. 5C) Representative ERG traces of rod (-1.7 log cd.s.m ² ), mixed rod-cone (0.51 log cd.s.m ² ) recorded dark adapted, and cone responses to single stimuli (0.51 log cd.s.m ² ) or 29-Hz cone flicker (0.26 log cd.s.m ² ) recorded light adapted at ~2 weeks after each of four light exposure sessions in a 77770-mutant dog injected in one eye with sc A A V 2/5 -RHOs _Ki -shRNA s2o (solid line) and with BSS (dashed line) in the contralateral eye. (FIG. 5D) Longitudinal quantification of maximal amplitudes of mixed rod- cone a- and b-waves (upper panel), and of cone lHz and 29Hz flicker responses (lower panel) in two 77770-mutant dogs injected in one eye with sc A A V 2/5- RHOsm-shRN A s2o (horizontal line shading) and in the contralateral eye with BSS (diagonal line shading) at similar time- points as shown in (FIG. 5C).

FIG. 6 shows a diagram of the transgene plasmid A AV -shRNAsio-RHOsio-

FIG. 7 shows an amino acid sequence of human rhodopsin illustrating sites of known mutations and deletions (based on review from Athanasiou et al.(l5)) and target sites of shRNAs and ribozyme knock down reagents evaluated in this current study.

FIG. 8 shows the digestion of human rhodopsin mRNA by hammerhead ribozyme 525 ( 7z525). Levels of wild type (white bar) or resistant/hardened (diagonal line shaded bar) 77770 transcripts measured by luciferase assay in HEK293 cells co-transfected with a plasmid expressing Rz525. Results were normalized to the same fusion transcript measured following co-transfection with a plasmid lacking ribozyme (black bar). Error bars represent standard error of the mean. * = P<0.05 for resistant/hardened 77770 relative to wild type 77770 by Student’s t test. FIGs. 9A-9C show the suppression of RHO expression with Rz525 in WT canine retinas. (FIG. 9A) Schematic representation of the retinas of RHO WT dogs injected subretinally with AAV2/5 -Rz525 at 20 or 50 xlO ¹¹ vg/mL titers showing the location of neuroretinal punches used for quantification of canine rhodopsin (RNA and protein) expression. Dashed lines, bleb boundaries; stippled area: tapetal region. (FIG. 9B)

Quantification of the levels of endogenous canine RHO RNA remaining in the treated (Tx) retinal area (injected with 50 xlO ¹¹ vg/mL titer) as a percentage of levels measured in the untreated (UnTx) area. (FIG. 9C) Representative immunoblot (upper) and quantification of the levels of endogenous canine RHO protein remaining (lower) in the treated retinal area as a percentage of levels measured in the untreated area of eyes injected with 20 or 50 xlO ¹¹ vg/mL titers. OSasOD designation refers to left eye being displayed as right eye for comparability.

FIGs. 10A-10E show the suppression of RHO expression with Rz525 in RHO mutant canine retinas. (FIG. 10A) Schematic representation of the retinas of RHO mutant dogs injected with BSS or AAV2/5 -Rz525 at 20 or 100 xlO ¹¹ vg/mL titers showing the location of neuroretinal punches used for quantification of canine rhodopsin (RNA and protein) expression. Dashed lines, bleb boundaries; stippled area: tapetal region. (FIG. 10B)

Quantification of the levels of endogenous canine RHO RNA remaining in the treated retinal area as a percentage of levels measured in the untreated area of eyes injected with BSS or AAV2/5 -Rz525 at 20 or 100 xlO ¹¹ vg/mL titers. (FIG. 10C) Representative immunoblot (upper) and quantification of the levels of endogenous canine RHO protein remaining (lower) in the treated retinal area as a percentage of levels measured in the untreated area of eyes injected with BSS or AAV2/5 -Rz525 at 20 or 100 xlO ¹¹ vg/mL titers. (FIG. 10D) ONL thickness topography two weeks post light exposure (post LE) in two RHO mutant dog eyes injected 8 weeks earlier with AAV2/5 -Rz525 at 20 or 100 xlO ¹¹ vg/mL titers. Dotted lines, bleb boundaries; dashed lines, ONL rescue boundaries. (FIG. 10E) Multifocal dark lesions (white arrows) visible by NIR cSLO imaging (upper and lower left panels) in both retinas of a RHO mutant dog treated 8 weeks earlier with AAV2/5 -Rz525 at 100 xlO ¹¹ vg/mL correspond on the OCT B scan (upper right panel) to focal nodules of increased reflectivity (white arrows) located in the subretinal space and RPE/choroid, and by histology (lower right panel) to focal inflammatory cell infiltrates. Retinal separation is visible on the OCT B scan (white arrowheads). Dashed lines, bleb boundaries; dotted line represents location of OCT B scan. OSasOD designation refers to left eye being displayed as right eye for comparability. FIGs. 11A-11C show inefficient suppression of RHO expression with shRNAm in WT canine retinas. (FIG. 11A) Schematic representation of the fundus of eyes of RHO WT dogs injected with scWY2/5-shRNAi _3i at 10 or 50 xlO ¹¹ vg/mL titers showing the location of neuroretinal punches used for quantification of canine rhodopsin (RNA and protein) expression at 8 weeks post-injection. Dashed lines, bleb boundaries; stippled area: tapetal region. (FIG. 11B) Quantification of the levels of endogenous canine RHO RNA remaining in the treated retinal area as a percentage of levels measured in the untreated area of eyes injected with 10 or 50 xlO ¹¹ vg/mL titers. (FIG. 11C) Representative immunoblot and quantification of the levels of endogenous canine RHO protein remaining in the treated retinal area as a percentage of levels measured in the untreated area of eyes injected with 10 or 50 xlO ¹¹ vg/mL titers. OSasOD designation refers to left eye being displayed as right eye for comparability.

FIG. 12 shows individual topographic results of all 10 RHO WT eyes injected with scAAY2/5-shRNA ₈₂₀ over a range of titers from lx to 50xl0 ⁿ vg/mL. ONL thickness (left), normalized IS/OS intensity (middle) and loci chosen within (squares on the right with surrounding dotted line) and outside (squares on the left) the bleb (right) for quantitation. 2194-OD and similar labels designate the individual animal and eye. All eyes are shown as equivalent right eyes to allow easier comparison. OSasOD designation refers to left eye being displayed as right eye for comparability.

FIGs. 13A-13G show suppression and replacement of rhodopsin with two separate vectors: incomplete protection of rods and retinal toxicity in RHO mutant eyes.

(FIGs. 13A-13C) OCT imaging and histological analysis of retinal protection against light exposure (LE) in a RHO mutant eye co-injected with 5xl0 ⁿ vg/mL of sc A A V 2/5-shRNA _H 2 _O and 5xl0 ⁿ vg/mL of sc A A V 2/5 -RHO sit ) (Treatment 1:1). (FIG. 13A) ONL thickness topography 7 weeks after injection/before light exposure (post Inj.) and four weeks post light exposure (post LE) Dotted lines, bleb boundaries; white arrow, location of OCT B scan shown in lower panel. (FIG. 13B) Rhodopsin (Rho)/human cone arrestin (hCA) co- immunolabeled retinal cryo section showing morphology of the outer nuclear layer (ONL) and outer segments (OS) in treated and untreated (UnTx) areas of the same eye shown in (FIG. 13A). (FIG. 13C) H&E stained retinal microphotograph taken within the treated area showing maximal rescue effect. (FIG. 13D-13F) Similar analysis as in (FIGs. 13A-13C) in a RHO mutant eye co-injected with 5xl0 ⁿ vg/mL of sc A A V 2/5-shRNA sin and 10 xlO ¹¹ vg/mL of scAAV2/5 -RHOsio (Treatment 1:2). (FIG. 13D) ONL thickness topography. Dotted lines, bleb boundaries; dashed lines, ONL rescue boundaries; white arrow corresponds to location of OCT B scan shown in lower panel; white arrows on OCT B scan show increased perivascular thickening and reflectivity. (FIG. 13E) Double fluorescence IHC (Rhodopsin; human cone arrestin). (FIG. 13F) H&E stained retinal microphotograph taken within the treated area showing limited ONL rescue and severe lesions of perivascular cell infiltration (arrows). (FIG. 13G) OCT imaging 11 weeks post injection in a RHO mutant eye co-injected with 5xl0 ⁿ vg/mL of cAAM2/5-shRNA, ₂ o and 10 xlO ¹¹ vg/mL of scAAV2/5 -RHOsio (Treatment 1:2) but that was not exposed to light. Dotted lines, bleb boundaries; grey and white arrows show locations of single OCT B scans (right panel). OSasOD designation refers to left eye being displayed as right eye for comparability.

FIGs. 14A-14D show suppression and replacement of rhodopsin with single vector prevents retinal degeneration in RHO mutant retinas (additional data to FIGs. 4A-4H). (FIG. 14A-14B) ONL thickness topography after injection/before light exposure (post Inj.) and two weeks post light exposure (post LE) in two RHO mutant dog eyes injected with scAAV2/5- RHOsio-shRNAsio at 5 xlO ¹¹ vg/mL titer. Dotted lines, bleb boundaries; dashed lines, ONL rescue boundaries. Insets, maps of significance showing retinal regions with ONL thickness (upper) and IS/OS intensity (lower) values compared point by point to the 99 ^th percentile confidence intervals of uninjected controls. (FIGs. 14C-14D) Rhodopsin (Rho)/human cone arrestin (hCA) co-immunolabeled retinal cryosections showing morphology of the outer nuclear layer (ONL) and outer segments (OS) in treated and untreated (UnTx) areas of the same eyes shown in (FIGs. 14A-14B). OSasOD designation refers to left eye being displayed as right eye for comparability.

FIGs. 15A-15C show the natural history of disease in RHO mutant (RHO ^t4r/+ ) dogs housed under different conditions of ambient illumination. (FIGs. 15A, 15B) Pseudocolor ONL topographies of representative dogs housed under cyclic dim-red light (FIG. 15 A) or cyclic standard white kennel illumination (FIG. 15B). All eyes are shown as equivalent right eyes. The optic nerve head (black circle) and major blood vessels (white lines), tapetum boundary (grey line), and fovea-like region ( ellipse) are overlaid (FIG. 15C) Schematic, loci selected in five retinal regions for quantitation of ONL thickness. C, central; ST,

superotemporal; SN, superonasal; IT, inf ero temporal; and IN, inferonasal. Plots of ONL thickness as a function of age at all sampled individual loci shown as a difference from the mean normal value at the same location. Dashed lines delimit 99 ^th percentile of normal variability.

FIGs. 16A-16E show recombinant AAV2/5 vector constructs used in the study. (FIG. 16A) Single- stranded AAV carrying a mouse opsin proximal promoter (mOP) driving the expression of a hammerhead ribozyme ( Rz525 ) designed to cleave murine, canine and human RHO mRNA. (FIG. 16B) Self-complementary AAV carrying an Hl RNA polymerase III promoter (Hl) driving the expression of a short hairpin RNA ( shRNA ) designed to cleave canine and human RHO mRNA. (FIG. 16C) Self complementary AAV carrying an Hl RNA polymerase III promoter (Hl) driving the expression of a short hairpin RNA ( shRNAsio ) designed to cleave canine and human RHO mRNA. (FIG. 16D) Self-complementary AAV carrying a human opsin promoter driving the expression of a replacement human RHO mRNA ( RHOsio ) designed to be resistant to suppression by shRNAsio . (FIG. 16E) Self complementary AAV carrying both the knockdown reagent shRNAsio and the human resistant replacement RHOsio . TR: AAV2 inverted terminal repeat; mTR: mutant TR; wtTR: wild type TR; SV40 SD/SA: splice donor/acceptor element derived from simian virus 40; hp Rz:

hairpin ribozyme; SV40 pA: SV40 derived polyadenylation terminal signal; HSV TK pro: human herpes simplex virus derived thymidine kinase promoter; Neo R: neomycin resistance gene. bGH pA: bovine growth hormone polyadenylation terminal signal; hGFP: humanized GFP.

FIG. 17 is an illustration of the experimental design of the long-term study of the AAV therapy in dogs. The ability of scAAN2/5-hOP-RHO820-Hl-shRNA820 to confer stable structural (assessed by OCT and IHC) and functional (assessed by ERG) protection of photoreceptors against repeated LE events that cause acute retinal degeneration in untreated RHO ^t4r/+ dog retinas, in four /7770-mutant dogs was evaluated. Each dog had its right eye (OD) subretinally-injected with 150 pL of the AAV construct at a titer of 5 x 10 ¹¹ vg/mL, while the contralateral left eye (OS) was subretinally-injected with a similar volume of balanced salt solution (BSS). PI: Post-Injection; LE: Light Exposure; OCT: Optical

Coherence Tomography; ERG: Electroretinography; IHC: Immunohistochemistry.

FIGs. 18A-18D show representative ERG traces of rod (-1.7 log cd.s.m ² , FIG. 18A), mixed rod-cone (0.51 log cd.s.m ² , FIG. 18B) recorded dark adapted, and cone responses to single stimuli (0.51 log cd.s.m ² , FIG. 18C) or 29-Hz cone flicker (0.26 log cd.s.m ² , FIG. 18D) recorded light adapted at ~2 weeks after each of four light exposure sessions in a 77770- mutant dog injected in one eye with scAAV2/5-hOP-/7/70S ^> 2O-H 1 -shRNA820 (dotted line) and with BSS (solid line) in the contralateral eye.

FIGs. 19A-19C show longitudinal quantification of maximal amplitudes of rod b- waves (FIG. 19A), mixed rod-cone a- and b-waves (FIG. 19B), and of cone lHz and 29Hz flicker responses (FIG. 19C) in four 77770-mutant dogs injected in one eye with scAAV2/5- RH0820-shRNA820 (horizontal line shading) and in the contralateral eye with BSS (diagonal line shading) at similar time-points as shown in FIGs. 18A-18D. AAV: scAAV2/5 -RHO820- shRNA820.

FIGs. 20A-20D show for each of the four RHO- mutant dogs outer nuclear layer (ONL) thickness maps and inner segment-outer segment (IS/OS) intensity maps prior to injection (pre-inj.), 12 weeks post- injection (before the first light exposure event), and 50 weeks post-injection (2 weeks after the fourth light exposure event).

FIG. 21 shows rhodopsin (RHO)/human cone arrestin (hCA) co-immunolabeled retinal cryosection from a RHO- mutant dog illustrating the morphology of the outer nuclear layer (ONL) and outer segments (OS) in untreated and treated areas of the same eye. The ONL thickness maps (lower image) shows the approximate location (asterisk) of the retinal untreated and treated areas shown in the upper image.

FIG. 22 shows OCT measurements of outer nuclear layer (ONL) thickness in mice treated with sc A A V 2/5- RHO, 2o-shRN A _IO or AAV2/5-GFP. P23H RHO C57B1/6 mice were treated with either AAV2/5-GFP or sc A A V 2/5 -RHOxio-shRNA , 2o i n one eye, and ONL thickness was analyzed at varying intervals: a) pre-treatment (“Pre”), b) 1 month post injection, c) 2 months post-injection, or d) 3 months post-injection.

FIG. 23 shows longitudinal quantification of maximal amplitudes of rod and mixed rod-cone a- and b-waves in P23H RHO mice injected in one eye with scAAV2/5 -RHO820- shRNA820 (circles and dashed line) or AAV2/5-GFP (squares and solid line). These two groups of mice were subjected to brief light flashes of varying intensities (-20 decibels (dB), - 10 dB and 0 dB). Electrical responses of these mice were analyzed at 1 month, 2 months, and 3 months post-injection.

FIG. 24 shows the sequences of most frequent short mRNAs expressed in HEK293 cells following transfection with sc A A V 2/5 -RHO820-shRNA 820, after size fractionation.

FIG. 25 shows the consensus sequence of the top“hits” of short mRNAs expressed in transfected HEK293 cells. As indicated by arrows, 52% of RNA molecules had 2 extra uridines at the 3’ end. The sequence of the 5’ end of the guide strand (UAG) in 73% of the RNA molecules was equivalent to what was predicted.

DETAILED DESCRIPTION

Aspects of the application provide methods and compositions that are useful for treating retinitis pigmentosa in a subject (e.g., in a subject having dominant retinitis pigmentosa such as a human subject having dominant retinitis pigmentosa). In some embodiments, a short hairpin RNA (shRNA) is provided that targets human, dog and/or mouse rhodopsin (RHO) mRNA. In some embodiments, the shRNA comprises a sense strand comprising the nucleotide sequence GUGGCAUUCUACAUCUUCA (SEQ ID NO: 1), an antisense strand comprising the nucleotide sequence

UGAAGAUGUAGAAUGCCAC (SEQ ID NO: 2), and a loop sequence. In some embodiments, the shRNA comprises a sense strand that comprises a nucleotide sequence that is one, two, or three nucleotides different from the sequence of SEQ ID NO: 1, and an antisense strand that comprises a nucleotide sequence that is one, two, or three nucleotides different from the sequence of SEQ ID NO: 2. In some embodiments, the loop comprises a sequence having a length of 5 to 10 nucleotides. In certain embodiments, the loop comprises a sequence having a length of 9 nucleotides. In some embodiments, the loop comprises ETETCAAGAGA (SEQ ID NO: 3). In certain embodiments, the loop is SEQ ID NO: 3. In certain embodiments, the loop comprises a nucleotide sequence that is one or two nucleotides different from the sequence of SEQ ID NO: 3.

In some embodiments, the shRNA comprises a sense strand comprising the nucleotide sequence GErGGCAUErCErACAUCErErCA (SEQ ID NO: 1), an antisense strand comprising the nucleotide sequence UGAAGAUGUAGAAUGCCAC (SEQ ID NO: 2), and a loop comprising the nucleotide sequence UUCAAGAGA (SEQ ID NO: 3). In some

embodiments, the shRNA comprises a sense strand comprising the nucleotide sequence GUGGCAUUCUACAUCUUCA (SEQ ID NO: 1), an antisense strand comprising the nucleotide sequence U GAAGAU GUAGAAU GCC ACUU (SEQ ID NO: 10), and a loop comprising the nucleotide sequence UUCAAGAGA (SEQ ID NO: 3). In some

embodiments, the shRNA comprises a sense strand that comprises a nucleotide sequence that is one, two, or three nucleotides different from the sequence of SEQ ID NO: 1 ; an antisense strand that comprises a nucleotide sequence that is one, two, or three nucleotides different from the sequence of SEQ ID NO: 10; and a loop that comprises a nucleotide sequence that is one or two nucleotides different from the sequence of SEQ ID NO: 3.

In some embodiments, the shRNA comprises or consists of the sequence of SEQ ID NO: 4 shown below. shRNA820 sequence:

GU GGC AUUCU AC AUCUU C AUUC A AG AG AU GAAGAU GU AG A AU GCC AC (SEQ ID NO: 4) In some embodiments, the shRNA comprises a nucleotide sequence that is one, two, or three nucleotides different from the sequence of SEQ ID NO: 4.

In certain embodiments, the shRNA comprises a sense and antisense strand comprising one of the following sets of sequences: i) a sense strand comprising the nucleotide sequence CUGCCUACAUGUUUCUGCU (SEQ ID NO: 21) and an antisense strand comprising the nucleotide sequence AGCAGAAACAUGETAGGCAG (SEQ ID NO: 22); ii) a sense strand comprising the nucleotide sequence CCETACAUGETErErCErGCErGAU (SEQ ID NO: 23) and an antisense strand comprising the nucleotide sequence

AUCAGCAGAAACAUGUAGG (SEQ ID NO: 24); iii) a sense strand comprising the nucleotide sequence GCAUGGUCAUCAUCAUGGU (SEQ ID NO: 25) and an antisense strand comprising the nucleotide sequence ACCAUGAUGAUGACCAUGC (SEQ ID NO: 26).

In other embodiments, the shRNA comprises: i) a sense strand comprising a nucleotide sequence that is one, two, or three nucleotides different from SEQ ID NO: 21, and an antisense strand comprising a nucleotide sequence that is one, two, or three nucleotides different from SEQ ID NO: 22; ii) a sense strand comprising a nucleotide sequence that is one, two, or three nucleotides different from SEQ ID NO: 23, and an antisense strand comprising a nucleotide sequence that is one, two, or three nucleotides different from SEQ ID NO: 24; or iii) a sense strand comprising a nucleotide sequence that is one, two, or three nucleotides different from SEQ ID NO: 25, and an antisense strand comprising a nucleotide sequence that is one, two, or three nucleotides different from SEQ ID NO: 26.

In some embodiments, the shRNA can be delivered using a vector as an shRNA driven by a promoter (e.g., a human Hl RNA promoter). In some embodiments, the vector is a plasmid. In some embodiments, the vector is a viral vector, such as an adeno-associated virus (AAV) vector. In some embodiments, the vector sequence encoding the shRNA comprises the sequence

GTGGCATTCTACATCTTCATTCAAGAGATGAAGATGTAGAATGCCAC (SEQ ID NO: 9). In some embodiments, the promoter driving shRNA expression comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence:

TAAAACGACGGCCAGTGAATTCATATTTGCATGTCGCTATGTGTTCTGGGAAATC ACC AT A A AC GTG A A ATGT CTTT GG ATTT GGG A AT CTT AT A AGTT CT GT AT GAG AC CACTCGGATCC (SEQ ID NO: 8). In some embodiments, the same vector comprises a coding sequence that encodes normal (e.g., wild-type) rhodopsin protein but is resistant to the action of the shRNA expressed by the vector.

In some embodiments, a normal (e.g., wild-type) rhodopsin (RHO) coding sequence that is hardened to an shRNA as described herein can have a sequence based on the human RHO gene (e.g., having a sequence shown in Accession No. NG_009l l5.l). In some embodiments, the normal RHO coding sequence is modified to include one or more (e.g., 2,

3, 4, 5, 6, 7, 8, 9, 10 or more) mutations that render the mRNA expressed by the coding sequence resistant to the shRNA as described herein. In some embodiments, the RHO coding sequence comprises the sequence GTGGCTTTTTATATATTCA (SEQ ID NO: 11) which may be resistant to an shRNA as described herein. In some embodiments, the RHO coding sequence comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 5 below.

RHO820 sequence:

CCAGCTGGAGCCCTGAGTGGCTGAGCTCAGGCCTTCGCAGCATTCTTGGGTGGGA

GCAGCCACGGGTCAGCCACAAGGGCCACAGCCATGAATGGCACAGAAGGCCCTA

ACTTCTACGTGCCCTTCTCCAATGCGACGGGTGTGGTACGCAGCCCCTTCGAGTA

CCCACAGTACTACCTGGCTGAGCCATGGCAGTTCTCCATGCTGGCCGCCTACATG

TTTCTGCTGATCGTGCTGGGCTTCCCCATCAACTTCCTCACGCTCTACGTCACCGT

CCAGCACAAGAAGCTGCGCACGCCTCTCAACTACATCCTGCTCAACCTAGCCGTG

GCTGACCTCTTCATGGTCCTAGGTGGCTTCACCAGCACCCTCTACACCTCTCTGCA

TGGATACTTCGTCTTCGGGCCCACAGGATGCAATTTGGAGGGCTTCTTTGCCACC

CTGGGCGGTGAAATTGCCCTGTGGTCCTTGGTGGTCCTGGCCATCGAGCGGTACG

TGGTGGTGTGTAAGCCCATGAGCAACTTCCGCTTCGGGGAGAACCATGCCATCAT

GGGCGTTGCCTTCACCTGGGTCATGGCGCTGGCCTGCGCCGCACCCCCACTCGCC

GGCTGGTCCAGGTACATCCCCGAGGGCCTGCAGTGCTCGTGTGGAATCGACTACT

ACACGCTCAAGCCGGAGGTCAACAACGAGTCTTTTGTCATCTACATGTTCGTGGT

CCACTTCACCATCCCCATGATTATCATCTTTTTCTGCTATGGGCAGCTCGTCTTCA

CCGTCAAGGAGGCCGCTGCCCAGCAGCAGGAGTCAGCCACCACACAGAAGGCA

GAGAAGGAGGTCACCCGCATGGTCATCATCATGGTCATCGCTTTCCTGATCTGCT

GGGTGCCCTACGCCAGCGTGGCTTTTTATATATTCACCCACCAGGGCTCCAACTT

CGGTCCCATCTTCATGACCATCCCAGCGTTCTTTGCCAAGAGCGCCGCCATCTAC

AACCCTGTCATCTTATCATGATGAACAAGCAGTTCCGGAACTGCATGCTCACCAC CATCTGCTGCGGCAAGAACCCACTGGGTGACGATGAGGCCTCTGCTACCGTGTCC AAGACGGAGACGAGCCAGGTGGCCCCGGCCTAAGACCTGCCTAGGACTCTGTGG CCGACTATAGGCGTCTCCCATCCCCTACACCTTCCCCCAGCCACAGCCATCCCAC CAG (SEQ ID NO: 5)

In some embodiments, the RHO coding sequence is driven by a promoter (e.g., a human opsin proximal promoter). In some embodiments, the promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:7 below.

Human opsin proximal promoter sequence:

CCTCATGGAGCTCCTCCTGTCAGAGGAGTGTGGGGACTGGATGACTCCAGAGGT

AACTTGTGGGGGAACGAACAGGTAAGGGGCTGTGTGACGAGATGAGAGACTGG

GAGAATAAACCAGAAAGTCTCTAGCTGTCCAGAGGACATAGCACAGAGGCCCAT

GGTCCCTATTTCAAACCCAGGCCACCAGACTGAGCTGGGACCTTGGGACAGACA

AGTCATGCAGAAGTTAGGGGACCTTCTCCTCCCTTTTCCTGGATCCTGAGTACCTC

TCCTCCCTGACCTCAGGCTTCCTCCTAGTGTCACCTTGGCCCCTCTTAGAAGCCAA

TTAGGCCCTCAGTTTCTGCAGCGGGGATTAATATGATTATGAACACCCCCAATCT

CCCAGATGCTGATTCAGCCAGGAGCTTAGGAGGGGGAGGTCACTTTATAAGGGT

CTGGGGGGGTCAGAACCCAGAGTCATCCAGCTGGAGCCCTGAGTGGCTGAGCTC

AGGCCTTCGCAGCATTCTTGGGTGGGAGCAGCCACGGGTCAGCCACA (SEQ ID

NO: 7)

In some embodiments, the vector as described herein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6 below. scANV2/5- iOP-RHO820-Hl-shRNA820 construct sequence:

CGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTT

TGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTC

CATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGT

AGCCATGCTCTAGGATCTGAATTCGGTACCCCTCATGGAGCTCCTCCTGTCAGAG GAGTGTGGGGACTGGATGACTCCAGAGGTAACTTGTGGGGGAACGAACAGGTAA

GGGGCTGTGTGACGAGATGAGAGACTGGGAGAATAAACCAGAAAGTCTCTAGCT

GTCCAGAGGACATAGCACAGAGGCCCATGGTCCCTATTTCAAACCCAGGCCACC

AGACTGAGCTGGGACCTTGGGACAGACAAGTCATGCAGAAGTTAGGGGACCTTC

TCCTCCCTTTTCCTGGATCCTGAGTACCTCTCCTCCCTGACCTCAGGCTTCCTCCT

AGTGTCACCTTGGCCCCTCTTAGAAGCCAATTAGGCCCTCAGTTTCTGCAGCGGG

GATTAATATGATTATGAACACCCCCAATCTCCCAGATGCTGATTCAGCCAGGAGC

TTAGGAGGGGGAGGTCACTTTATAAGGGTCTGGGGGGGTCAGAACCCAGAGTCA

TCCAGCTGGAGCCCTGAGTGGCTGAGCTCAGGCCTTCGCAGCATTCTTGGGTGGG

AGCAGCCACGGGTCAGCCACATCTAGAGGATCCGGTACTCGAGGAACTGAAAAA

CCAGAAAGTTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCGGATC

CGGTGGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTA

GGCCTGTACGGAAGTGTTACTTCTGCTCTAAAAGCTGCGGAATTGTACCCGCGGC

CGCCCAGCTGGAGCCCTGAGTGGCTGAGCTCAGGCCTTCGCAGCATTCTTGGGTG

GGAGCAGCCACGGGTCAGCCACAAGGGCCACAGCCATGAATGGCACAGAAGGC

CCTAACTTCTACGTGCCCTTCTCCAATGCGACGGGTGTGGTACGCAGCCCCTTCG

AGTACCCACAGTACTACCTGGCTGAGCCATGGCAGTTCTCCATGCTGGCCGCCTA

CATGTTTCTGCTGATCGTGCTGGGCTTCCCCATCAACTTCCTCACGCTCTACGTCA

CCGTCCAGCACAAGAAGCTGCGCACGCCTCTCAACTACATCCTGCTCAACCTAGC

CGTGGCTGACCTCTTCATGGTCCTAGGTGGCTTCACCAGCACCCTCTACACCTCTC

TGCATGGATACTTCGTCTTCGGGCCCACAGGATGCAATTTGGAGGGCTTCTTTGC

CACCCTGGGCGGTGAAATTGCCCTGTGGTCCTTGGTGGTCCTGGCCATCGAGCGG

TACGTGGTGGTGTGTAAGCCCATGAGCAACTTCCGCTTCGGGGAGAACCATGCCA

TCATGGGCGTTGCCTTCACCTGGGTCATGGCGCTGGCCTGCGCCGCACCCCCACT

CGCCGGCTGGTCCAGGTACATCCCCGAGGGCCTGCAGTGCTCGTGTGGAATCGA

CTACTACACGCTCAAGCCGGAGGTCAACAACGAGTCTTTTGTCATCTACATGTTC

GTGGTCCACTTCACCATCCCCATGATTATCATCTTTTTCTGCTATGGGCAGCTCGT

CTTCACCGTCAAGGAGGCCGCTGCCCAGCAGCAGGAGTCAGCCACCACACAGAA

GGCAGAGAAGGAGGTCACCCGCATGGTCATCATCATGGTCATCGCTTTCCTGATC

TGCTGGGTGCCCTACGCCAGCGTGGCTTTTTATATATTCACCCACCAGGGCTCCA

ACTTCGGTCCCATCTTCATGACCATCCCAGCGTTCTTTGCCAAGAGCGCCGCCAT

CTACAACCCTGTCATCTATATCATGATGAACAAGCAGTTCCGGAACTGCATGCTC

ACCACCATCTGCTGCGGCAAGAACCCACTGGGTGACGATGAGGCCTCTGCTACC

GTGTCCAAGACGGAGACGAGCCAGGTGGCCCCGGCCTAAGACCTGCCTAGGACT CTGTGGCCGACTATAGGCGTCTCCCATCCCCTACACCTTCCCCCAGCCACAGCCA

TCCCACCAGGCGGCCGCGGGGATCCAGACATGATAAGATACATTGATGAGTTTG

GACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTG

ATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAA

CAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAGT

CGACTAAAACGACGGCCAGTGAATTCATATTTGCATGTCGCTATGTGTTCTGGGA

AATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATG

AGACCACTCGGATCCGTGGCATTCTACATCTTCATTCAAGAGATGAAGATGTAGA

ATGCCACTTTTTAAGCTTTTTGGCGTAATCATGGTCGACATTGGATCGGATCCCG

GGCCCGTCGACTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGC

CATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCC

ACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTC

ATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAG

ACAATAGCAGGAACCCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGG

GCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGA

GCGAGCGCGCAGCTGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGT

TTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGT

TCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCAC

AGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGG

CCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCC

TGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGG

ACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTT

CCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGG

CGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCC

AAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCG

GTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGC

AGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTT

CTTG A AGT GGT GGCCT A ACT AC GGCT AC ACT AG A AGG AC AGT ATTT GGT AT CT GC

GCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCA

AACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCG

CAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCT

CAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGG

ATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTA

TATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTAT CTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGA

TAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCG

AGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAG

GGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAAT

TGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTG

TTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTC

AGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAA

AAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGT

GTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCG

TAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTG

TATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCA

CATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAA

CTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCAC

CCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAAC

AGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAA

TACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTC

ATG AGC GG AT AC AT ATTT G A ATGT ATTT AG A A A A AT A A AC A A AT AGGGGTT CC G

CGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGA

CATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGG

TGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTG

TCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGT

TGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAG

AGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCG

CATCAGGAAATCCAACATCCAATAAATCATACAGGCAAGGCAAAGAATTAGCAA

AATTAAGCAATAAAGCCTCAGAGCATAAAGCTAAATCGGTTGTACCAAAAACAT

TATGACCCTGTAATACTTTTGCGGGAGAAGCCTTTATTTCAACGCAAGGATAAAA

_{ATTTTTAGAACCCTCATATATTTTAAATGCAATGCCTGAGTAATGTGTAGGTAAA}

GATTCAAACGGGTGAGAAAGGCCGGAGACAGTCAAATCACCATCAATATGATAT

TC A ACC GTT CT AGCT GAT A A ATTC AT GCC GG AG AGGGT AGCT ATTTTT G AG AGGT

CTCTACAAAGGCTATCAGGTCATTGCCTGAGAGTCTGGAGCAAACAAGAGAATC

GATGAACGGTAATCGTAAAACTAGCATGTCAATCATATGTACCCCGGTTGATAAT

CAGAAAAGCCCCAAAAACAGGAAGATTGTATAAGCAAATATTTAAATTGTAAAC

GTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAA

CCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGAT AGGGTT G AGT GTT GTT CC AGTTTGG A AC A AG AGT CC ACT ATT A A AG A AC GT GG AC

TCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAA

CCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGA

ACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGG

CGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGT

GTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTAC

AGGGCGCGTACTATGGTTGCTTTGACGAGCACGTATAACGTGCTTTCCTCGTTAG

AATCAGAGCGGGAGCTAAACAGGAGGCCGATTAAAGGGATTTTAGACAGGAAC

GGTACGCCAGAATCCTGAGAAGTGTTTTTATAATCAGTGAGGCCACCGAGTAAA

AGAGTCTGTCCATCACGCAAATTAACCGTTGTCGCAATACTTCTTTGATTAGTAA

TAACATCACTTGCCTGAGTAGAAGAACTCAAACTATCGGCCTTGCTGGTAATATC

CAGAACAATATTACCGCCAGCCATTGCAACAGGAAAAACGCTCATGGAAATACC

TACATTTTGACGCTCAATCGTCTGGAAATCCATTCGCCATTCAGGCTGCGCAACT

GTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCG (SEQ

ID NO: 6)

Aspects of the disclosure relate to recombinant adeno-associated virus (rAAV) particles for delivery of an rAAV vector as described herein (e.g., encoding an shRNA and/or a replacement RHO) into various tissues, organs, and/or cells. In some embodiments, the rAAV particles comprise a capsid protein as described herein, e.g., an AAV5 capsid protein. In some embodiments, the vector contained within the rAAV particle encodes an RNA of interest (e.g., an shRNA comprising the sequence of SEQ ID NO: 4) and comprises a replacement RHO coding sequence (e.g., comprising the sequence of SEQ ID NO: 5).

Recombinant AAV (rAAV) vectors contained within an rAAV particle may comprise at a minimum (a) one or more heterologous nucleic acid regions (e.g., encoding an shRNA and/or a RHO protein) and (b) one or more regions comprising inverted terminal repeat (ITR) sequences (e.g., wild-type ITR sequences or engineered ITR sequences) flanking the one or more heterologous nucleic acid regions. In some embodiments, the heterologous nucleic acid region encodes an RNA of interest (e.g., an shRNA comprising the sequence of SEQ ID NO: 4) and comprises a replacement RHO coding sequence (e.g., comprising the sequence of SEQ ID NO: 5). In some embodiments, the rAAV vector is between 4kb and 5kb in size (e.g., 4.2 to 4.7 kb in size). This rAAV vector may be encapsidated by a viral capsid, such as an AAV5 capsid. In some embodiments, the rAAV vector is single- stranded. In some embodiments, the rAAV vector is double- stranded. In some embodiments, a double- stranded rAAV vector may be, for example, a self-complementary vector that contains a region of the vector that is complementary to another region of the vector, initiating the formation of the double-strandedness of the vector.

The rAAV particle may be of any AAV serotype, including any derivative or pseudotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2/1, 2/5, 2/8, or 2/9). As used herein, the serotype of an rAAV particle refers to the serotype of the capsid proteins. In some embodiments, the rAAV particle is AAV5. Non-limiting examples of derivatives and pseudotypes include rAAV2/l, rAAV2/5, rAAV2/8, rAAV2/9, AAV2-AAV3 hybrid, AAVrh.lO, AAVhu.l4, AAV3a/3b, AAVrh32.33, AAV-HSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15/17, AAVM41, AAV9.45, AAV6(Y445F/Y731F), AAV2.5T, AAV-HAE1/2, AAV clone 32/83, AAVShHlO, AAV2 (Y->F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, and AAVr3.45. Such AAV serotypes and derivatives/pseudotypes, and methods of producing such

derivatives/pseudotypes are known in the art (see, e.g., Mol Ther. 2012 Apr;20(4):699-708. doi: 10. l038/mt.20l 1.287. Epub 2012 Jan 24. The AAV vector toolkit: poised at the clinical crossroads. Asokan Al, Schaffer DV, Samulski RJ.). In some embodiments, the rAAV particle is a pseudotyped rAAV particle, which comprises (a) a nucleic acid vector comprising ITRs from one serotype (e.g., AAV2) and (b) a capsid comprised of capsid proteins derived from another serotype (e.g., AAV5). Methods for producing and using pseudotyped rAAV vectors are known in the art (see, e.g., Duan et ah, J. Virol., 75:7662- 7671, 2001; Halbert et ah, J. Virol., 74:1524-1532, 2000; Zolotukhin et ah, Methods, 28:158- 167, 2002; and Auricchio et ah, Hum. Molec. Genet., 10:3075-3081, 2001).

Methods of producing rAAV particles and rAAV vectors are also known in the art and commercially available (see, e.g., Zolotukhin et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2002) 158-167; and U.S. Patent Publication Numbers US 2007/0015238 and US20120322861, which are incorporated herein by reference; and plasmids and kits available from ATCC and Cell Biolabs, Inc.). For example, a plasmid containing the rAAV vector may be combined with one or more helper plasmids, e.g., that contain a rep gene (e.g., encoding Rep78, Rep68, Rep52 and Rep40) and a cap gene (e.g., encoding VP1, VP2, and VP3, including a modified VP3 region as described herein), and transfected into a producer cell line such that the rAAV particle can be packaged and subsequently purified.

In some embodiments, the one or more helper plasmids include a first helper plasmid comprising a rep gene and a cap gene (e.g., encoding a rAAV capsid protein as described herein) and a second helper plasmid comprising a Ela gene, a Elb gene, a E4 gene, a E2a gene, and a VA gene. In some embodiments, the rep gene is a rep gene derived from AAV2 or AAV5 and the cap gene is derived from AAV2 or AAV5 and may include modifications to the gene in order to produce the modified capsid protein described herein. Helper plasmids, and methods of making such plasmids, are known in the art and commercially available (see, e.g., pDM, pDG, pDPlrs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E/R585E), and pDP8.ape plasmids from PlasmidFactory, Bielefeld, Germany; other products and services available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA;

Agilent Technologies, Santa Clara, Ca; and Addgene, Cambridge, MA; pxx6; Grimm el al. (1998), Novel Tools for Production and Purification of Recombinant Adenoassociated Virus Vectors, Human Gene Therapy , Vol. 9, 2745-2760; Kem, A. et al. (2003), Identification of a Heparin-Binding Motif on Adeno- Associated Virus Type 2 Capsids, Journal of Virology,

Vol. 77, 11072-11081.; Grimm et al. (2003), Helper Virus-Free, Optically Controllable, and Two-Plasmid-Based Production of Adeno-associated Virus Vectors of Serotypes 1 to 6, Molecular Therapy, Vol. 7, 839-850; Kronenberg et al. (2005), A Conformational Change in the Adeno-Associated Virus Type 2 Capsid Leads to the Exposure of Hidden VP1 N Termini, Journal of Virology, Vol. 79, 5296-5303; and Moullier, P. and Snyder, R.O. (2008),

International efforts for recombinant adeno-associated viral vector reference standards, Molecular Therapy, V ol. 16, 1185-1188).

An exemplary, non-limiting, rAAV particle production method is described next.

One or more helper plasmids are produced or obtained, which comprise rep and cap ORFs for the desired AAV serotype and the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. The cap ORF may also comprise one or more modifications to produce a modified capsid protein as described herein. HEK293 cells (available from ATCC®) are transfected via CaP04-mediated transfection, lipids or polymeric molecules such as Polyethylenimine (PEI) with the helper plasmid(s) and a plasmid containing a nucleic acid vector described herein. The HEK293 cells are then incubated for at least 60 hours to allow for rAAV particle production. Alternatively, in another example Sf9-based producer stable cell lines are infected with a single recombinant baculovirus containing the nucleic acid vector. As a further alternative, in another example HEK293 or BHK cell lines are infected with a HSV containing the nucleic acid vector and optionally one or more helper HSVs containing rep and cap ORFs as described herein and the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. The HEK293, BHK, or Sf9 cells are then incubated for at least 60 hours to allow for rAAV particle production. The rAAV particles can then be purified using any method known the art or described herein, e.g., by iodixanol step gradient, CsCl gradient, chromatography, or polyethylene glycol (PEG) precipitation.

The disclosure also contemplates host cells that comprise an shRNA, a vector, or an rAAV particle as described herein. Such host cells include mammalian host cells, with human host cells being preferred, and may be isolated, e.g., in cell or tissue culture. In some embodiments, the host cell is a cell of the eye.

The disclosure also contemplates host cells that comprise an shRNA, a vector, an rAAV particle, and the mRNA expressed after infection of the host cell by the rAAV particles described herein or transfection by the constructs described herein. In certain embodiments, the host cells provided herein comprise short mRNA sequences that are different from those found in nature. In certain embodiments, the host cells comprise short mRNA sequences having at least 95% or 99.5% sequence identity with any one of SEQ ID NOs: 40-46. The host cells may comprise short mRNA sequences comprising the sequence of any one of SEQ ID NOs: 40-46. Such host cells include mammalian host cells, with human host cells being preferred, and may be isolated, e.g., in cell or tissue culture. In some embodiments, the host cell is a cell of the eye.

Exemplary mammalian cells include human cells, rodent cells and canine cells. In some embodiments, the mammalian cells are derived from a human (e.g., a human having or known to have, for example diagnosed as having, retinitis pigmentosa, for example dominant retinitis pigmentosa).

In some embodiments, a composition is provided which comprises an shRNA, a vector, or an rAAV particle as described herein and optionally a pharmaceutically acceptable carrier. In some embodiments, the compositions described herein can be administered to a subject in need of treatment. In some embodiments, the subject has or is suspected of having one or more conditions, diseases, or disorders of the brain and/or eye (e.g., retinitis pigmentosa such as dominant retinitis pigmentosa). In some embodiments, the subject has or is suspected of having one or more of the conditions, diseases, and disorders disclosed herein (e.g., retinitis pigmentosa such as dominant retinitis pigmentosa). In some embodiments, the subject has one or more endogenous mutant rho alleles (e.g., associated with or that cause a disease or disorder of the eye or retina). In some embodiments, the subject has at least one dominant mutant rho allele (e.g., that causes dominant retinitis pigmentosa). In some embodiments, the subject is a human. In some embodiments, the subject is a non-human primate. Non-limiting examples of non-human primate subjects include macaques (e.g., cynomolgus or rhesus macaques), marmosets, tamarins, spider monkeys, owl monkeys, vervet monkeys, squirrel monkeys, baboons, gorillas, chimpanzees, and orangutans. Other exemplary subjects include domesticated animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters.

In some embodiments, the dose of rAAV particles administered to a cell or a subject may be on the order ranging from 10 ⁶ to 10 ¹⁴ particles/mL or 10 ³ to 10 ¹⁵ particles/mL, or any values therebetween for either range, such as for example, about 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , or 10 ¹⁴ particles/mL. In one embodiment, rAAV particles of higher than 10 ¹³ particles/mL are be administered. In some embodiments, the dose of rAAV particles administered to a subject may be on the order ranging from 10 ⁶ to 10 ¹⁴ vector genomes(vgs)/mL or 10 ³ to 10 ¹⁵ vgs/mL, or any values therebetween for either range, such as for example, about 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , or 10 ¹⁴ vgs/mL. In one embodiment, rAAV particles of higher than 10 ¹³ vgs/mL are be administered. The rAAV particles can be administered as a single dose, or divided into two or more administrations as may be required to achieve therapy of the particular disease or disorder being treated. In some embodiments, 0.0001 mL to 10 mLs (e.g., 0.0001 mL, 0.001 mL, 0.01 mL, 0.1 mL, 1 mL, 10 mLs) are delivered to a subject in a dose.

In some embodiments, rAAV particle titers range from 1 x l0 ¹⁰ -5 x 10 ¹³ vg/ml. In some embodiments, rAAV particle titers can be 1 x 10 ¹⁰ , 2.5 x 10 ¹⁰ , 5 x 10 ¹⁰ , 1 x 10 ¹¹ , 2.5 x 10 ¹¹ , 5 x 10 ¹¹ , 1 x 10 ¹² , 2.5 x 10 ¹² , 5 x 10 ¹² , 1 x 10 ¹³ , 2.5 x 10 ¹³ , or 5 x 10 ¹³ vg/mL. In some embodiments, particle titers are less than 1 x 10 ¹⁰ vg/mL. In some embodiments, rAAV particle titers are greater than 1 x 10 ¹⁵ vg/mL. In one embodiment, rAAV particle titers are greater than 5 x 10 ¹³ vgs/mL. In some embodiments, rAAV particles are administered via methods further described herein (e.g., subretinally or intravitreally).

The rAAV particles can be administered as a single dose, or divided into two or more administrations as may be required to achieve therapy of the particular disease or disorder being treated. In some embodiments, the rAAV particles are administered over a period of days or weeks. In some embodiments, from 1 to 500 microliters of a composition (e.g., comprising an rAAV particle) described in this application is administered to one or both eyes of a subject. For example, in some embodiments, about 1, about 10, about 50, about 100, about 200, about 300, about 400, or about 500 microliters can be administered to each eye. However, it should be appreciated that smaller or larger volumes could be administered in some embodiments. In some embodiments, the rAAV particles, compositions and methods of treatment disclosed herein preserve the integrity of the structure of rod photoreceptors in the subject, preserve ONL thickness and/or confer protection from degeneration of at least about 12 weeks, at least about 18 weeks, at least about 24 weeks, at least about 30 weeks, at least about 36 weeks, at least about 42 weeks, at least about 48 weeks, at least about 54 weeks, or at least about 60 weeks for retinal structure and function in the subject following a single administration to the eye.

In some embodiments, the disclosure provides formulations of one or more rAAV- based 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, and in particular, for therapy of human cells, tissues, and diseases affecting man.

If desired, rAAV particle or nucleic acid vectors 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 therapeutic polypeptides, biologically active fragments, or variants thereof. 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 rAAV particles 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.

Formulation of pharmaceutically-acceptable excipients and carrier solutions is well- known 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, intra- articular, and intramuscular administration and formulation.

Typically, these formulations may contain at least about 0.1% of the therapeutic agent (e.g., rAAV particle) 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 70% or 80% or more of the weight or volume of the total formulation. Naturally, the amount of therapeutic agent(s) (e.g., rAAV particle) in each therapeutically-useful composition may be prepared in 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 skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

In certain circumstances it will be desirable to deliver an shRNA, a vector, or an rAAV particle as described herein in suitably formulated pharmaceutical compositions disclosed herein either subcutaneously, intraocularly, intravitreally, parenterally,

subcutaneously, intravenously, intracerebro-ventricularly, intramuscularly, intrathecally, orally, intraperitoneally, by oral or nasal inhalation, or by direct injection to one or more cells, tissues, or organs by direct injection.

The pharmaceutical forms of compositions (e.g., comprising an shRNA, a vector, or an rAAV particle as described herein) suitable for injectable use include sterile aqueous solutions or dispersions. In some embodiments, the form is sterile and fluid to the extent that easy syringability exists. In some embodiments, the form is stable under the conditions of manufacture and storage and is preserved against the contaminating action of

microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, saline, 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 term“carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the shRNA, vector, or rAAV particle as described herein is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum oil such as mineral oil, vegetable oil such as peanut oil, soybean oil, and sesame oil, animal oil, or oil of synthetic origin. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers.

The compositions of the present disclosure can be delivered to the eye through a variety of routes. They may be delivered intraocularly, by topical application to the eye or by intraocular injection into, for example the vitreous (intravitreal injection) or subretinal (subretinal injection) inter-photoreceptor space. In some embodiments, they are delivered to rod photoreceptor cells. Alternatively, they may be delivered locally by insertion or injection into the tissue surrounding the eye. They may be delivered systemically through an oral route or by subcutaneous, intravenous or intramuscular injection. Alternatively, they may be delivered by means of a catheter or by means of an implant, wherein such an implant is made of a porous, non-porous or gelatinous material, including membranes such as silastic membranes or fibers, biodegradable polymers, or proteinaceous material. They can be administered prior to the onset of the condition, to prevent its occurrence, for example, during surgery on the eye, or immediately after the onset of the pathological condition or during the occurrence of an acute or protracted condition.

For administration of an injectable aqueous solution, for example, the solution may 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, intravitreal, 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 NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" l5th Edition, 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, e.g., FDA Office of Biologies standards.

Sterile injectable solutions may be prepared by incorporating an shRNA, a vector, or an rAAV particle as described herein in the required amount in the appropriate solvent with several of the other ingredients enumerated above, 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.

The amount of composition (e.g., comprising an shRNA, a vector, or an rAAV particle as described herein) and time of administration of such composition will be within the purview of the skilled artisan having benefit of the present teachings. It is likely, however, that the administration of therapeutically-effective amounts of the disclosed compositions may be achieved by a single administration, such as for example, a single injection of sufficient numbers of rAAV particles to provide therapeutic benefit to the patient undergoing such treatment. Alternatively, in some circumstances, it may be desirable to provide multiple, or successive administrations of the composition, either over a relatively short, or a relatively prolonged period of time, as may be determined by the medical practitioner overseeing the administration of such compositions.

In some embodiments, rod cells remain structurally intact and/or viable upon silencing of cellular rhodopsin gene expression. In some embodiments, rods cells in which cellular rhodopsin gene expression is silenced have shortened outer segments which would normally contain rhodopsin. In some embodiments, the length of the outer segments can be maintained or restored (e.g., partially or completely) using the exogenously added (hardened) rhodopsin gene, the expression of which is resistant to silencing using the compositions described in this application. In some embodiments, administration of a composition described herein to a subject having retinitis pigmentosa (e.g., dominant retinitis pigmentosa) preserves the integrity of the structure of rod photoreceptors in the subject, preserves ONL thickness and/or confers protection from degeneration of at least about 12 weeks, at least about 18 weeks, at least about 24 weeks, at least about 30 weeks, at least about 36 weeks, at least about 42 weeks or at least about 48 weeks, at least about 54 weeks, or at least about 60 weeks for retinal structure and function in the subject.

To“treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject (e.g., retinitis pigmentosa). The compositions described above are typically administered to a subject in an effective amount, that is, an amount capable of producing a desirable result.

The desirable result will depend upon the active agent being administered. For example, an effective amount of a rAAV particle may be an amount of the particle that is capable of transferring a heterologous nucleic acid to a host organ, tissue, or cell.

Toxicity and efficacy of the compositions utilized in methods of the disclosure can be determined by standard pharmaceutical procedures, using either cells in culture or experimental animals to determine the LD50 (the dose lethal to 50% of the population). The dose ratio between toxicity and efficacy the therapeutic index and it can be expressed as the ratio LD50/ED50. Those compositions that exhibit large therapeutic indices are preferred. While those that exhibit toxic side effects may be used, care should be taken to design a delivery system that minimizes the potential damage of such side effects. The dosage of compositions as described herein lies generally within a range that includes an ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present disclosure to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

EXAMPLES

Example 1

Inherited retinal degenerations are caused by mutations in > 250 genes that affect photoreceptor cells or the retinal pigment epithelium and cause vision loss. For autosomal recessive and X-linked retinal degenerations, significant progress has been achieved in the field of gene therapy as evidenced by the growing number of clinical trials, and the recent commercialization of the first gene therapy for a form of congenital blindness. However, in spite of significant efforts to develop a treatment for the most common form of autosomal dominant retinitis pigmentosa (adRP) caused by >150 mutations in the rhodopsin ( RHO ) gene, translation to the clinic has stalled. Here, a highly efficient novel short hairpin RNA (shRNA) was first identified that targets human (and canine) RHO in a mutation-independent manner. In a single adeno-associated viral (AAV) vector, this shRNA was combined with a human RHO replacement cDNA made resistant to RNA interference, and this construct (referred to hereafter as“scAPYfUS-RHOsio-shRNAsio”, see FIG. 6) was tested in a naturally- occurring canine model of RHO-adRP. Subretinal vector injections led to near complete suppression of endogenous canine RHO RNA while the human RHO replacement cDNA resulted in up to 30% of normal RHO protein levels. Non-invasive retinal imaging showed complete protection of photoreceptors from retinal degeneration in treated areas.

Histopathology confirmed retention of normal photoreceptor structure and RHO expression in rod outer segments. Long-term (> 8 months) follow-up by retinal imaging and

electroretinography indicated stable structural and functional preservation. Efficacy of this gene therapy in a clinically relevant large animal model paves the way for treating patients with RHO-adRP.

The past two decades have seen a steep rise in the number of gene therapies entering clinical trials(l, 2) and in recent years a small number of them have received marketing approval by regulatory authorities in China, Europe and the US. (3) The vast majority of these trials have targeted cancer, cardiovascular, and inherited monogenic diseases. (1) Strategies for inherited monogenic diseases are by necessity based on the mechanism of disease. For the vast majority of loss of function mutations, the strategy is gene augmentation. (4) For mutations that cause a dominant-negative effect, gene augmentation may also provide some therapeutic benefit by diluting the deleterious effect of the mutant product.(5, 6) However, in the case of mutations that confer a toxic gain-of-function, strategies that are being

investigated include ablation of the gene or correction of the defect at the DNA level (e.g. CRISPR/Cas9 gene editing), transcriptional repression, and RNA knockdown/suppression. (7, 8)

Mutations in more than 250 genes are known to cause inherited retinal diseases (sph.uth.edu/retnet/), and considerable advances have been made in gene therapy approaches because of the accessibility of the retina. Clinical trials of gene augmentation are currently ongoing for at least six autosomal recessive, three X-linked, and one maternally-inherited mitochondrial retinal diseases. (9) There are no trials for autosomal dominant retinal diseases, the most common of which is autosomal dominant retinitis pigmentosa (adRP) caused by mutations in the rhodopsin ( RHO ) gene. (10-14) For the more than 150 identified RHO mutations, several putative pathogenic mechanisms based mostly on in vitro findings have been proposed (for reviews see 15, 16), yet detailed characterization of RHO-adRP patient phenotype is consistent with two major categories. (17-19) Class A mutant patients have severe loss of rods from early life, and realistic therapeutic approaches should be directed at prolonging cone survival. On the other hand, patients with Class B mutants can have rods that survive for decades into late adult life in some retinal regions or throughout the retina, and could benefit from a gene therapy aimed at rescuing the remaining rods and preventing secondary cone cell loss. (20)

Over the past 20 years, efforts on gene therapy for RHO-adRP have focused on either reducing expression of specific mutant alleles(2l-28), or developing a mutation-independent strategy. The latter strategy combines knocking down the expression of both the mutant and wild type (WT) RHO proteins(29-39) while providing as replacement a resistant RHO cDNA that encodes the WT protein. (40-43) Resistance is conferred by codon modification at degenerate/wobble nucleotides within the target site, which prevents hybridization with the knockdown reagent. Such mutation-independent“knockdown and replacement” strategy aims at addressing the high allelic heterogeneity in RHO-adRP, while circumventing the technical and financial challenges that would be inherent in developing multiple gene therapies for individual RHO mutations. The retinal co-delivery of the two components using either two separate(42), or a single AAV vector (41, 43) have been explored in transgenic mice by separate research groups. However complete prevention or arrest of the ongoing rod degeneration was not achieved.

Here, a highly effective short hairpin RNA, shRNAxio, was identified that targets human RHO in a mutation-independent manner. When combined with a resistant form of human RHO, and co-packaged in a single recombinant adeno-associated viral (AAV) particle, this construct with dual knockdown and replacement functions provided long-term protection against retinal degeneration in a naturally-occurring canine model of R/70-adRP.

Results

Optimal suppression of wild-type Rhodopsin with shRNAsio

Four knockdown reagents, including a previously identified(33) hammerhead ribozyme ( Rz525 ), and three novel shRNAs ( shRNA , S1IRNA _B4 , shRNAsio) that target distinct homologous regions of canine and human RHO (FIG. 7), were screened initially using in vitro assays. Silencing of RHO expression was very effective with Rz525 both in vitro (FIG. 8), and in WT (FIGs. 9A-9C; Table 1, group C) and ///70-mutant canine eyes (FIGs.lOA-lOC; Table 1, group F). However, due to severe retinal complications associated with the high viral titers of AAV2/5 -Rz525 needed to achieve near complete suppression of RHO expression (Results below and FIGs. 10D-10E), further development of Rz525 was discontinued. In vitro screening of shRNAs showed that shRNAm resulted in only ~ 50% reduction of WT human RHO protein (FIGs. 1A, 1B), and failed to suppress mutant human RHO P23H (FIGs. 1C, 1D) and T17M (FIGs. 1E, 1F). Moreover, there was limited suppression of RHO expression in injected canine WT eyes ( Results below, FIGs. 11A-11C, Table 1, group B).

The most potent shRNA to suppress expression of both WT and mutant (P23H and T17M) human RHO protein in vitro was shRNAsio (FIGs. 1A-1F). In parallel, codon- modified form of human RHO, RHOsio , that contained four altered nucleotides at

degenerate/wobble positions within the target site of shRNAsio was confirmed to be resistant to shRNAsio suppression (FIGs. 1G, 1H). Once confirmed that shRNAsio targeted RHO in a mutation-independent manner, it was selected as the lead knockdown reagent for further evaluation in WT and ///70-mutant dogs.

Validation of shRNAsio was performed first in WT dogs to determine the titer at which RHO expression can be substantially reduced with expected changes occurring only in outer segments, where RHO is a major signaling and structural protein, but without major stress or degeneration of the remaining cellular compartments of rod photoreceptors. Subretinal injections were performed in ten WT canine eyes with AAV -shRNA 820 titers ranging from lx to 50xl0 ^u vg/mL (Table 1, group A). Treated eyes were evaluated at 7-8 weeks post-injection by in-life spectral-domain optical coherence tomography (OCT) imaging of the retinal structure and compared to uninjected control eyes. In a representative uninjected WT eye, cross-sectional imaging in the superior retina with OCT revealed hypo- and hyper-scattering layers corresponding to different retinal lamina (FIG. 2A, left). (44) The thickness of the outer nuclear layer (ONL) where the photoreceptor nuclei reside, and the backscatter intensity originating near the inner segment-outer segment (IS/OS) junction were of primary interest to this study (FIG. 2A, left). IS/OS intensity is expected to be sensitive to changes in outer segment length, alignment and spatial density, and thus can be used as a non-invasive surrogate measure of outer segment health. The normalized IS/OS intensity topography of the uninjected WT tends to be uniform (FIG. 2A, middle column). ONL thickness topography in the uninjected WT eye was also relatively homogeneous with incrementally greater values in the central retina supero-temporal to the optic nerve head (ONH), and incrementally smaller values in the non-tapetal areas of superior and inferior retina (FIG. 2A, right column). Lower and intermediate titer injections represented by eyes injected with vector at lxlO ¹¹ vg/mL (FIG. 2B), or 5xl0 ^u vg/mL (FIG. 2C) showed no qualitative structural changes between the injected and neighboring uninjected regions.

To define the optimal titer at which structural consequences of RHO knockdown are detectable but mild, retinal locations were systematically sampled (FIG. 12). A great majority of the injected locations for the two lowest titers (lx and 2.5xlO ⁿ vg/mL) were comparable to uninjected control eyes with respect to IS/OS intensity and ONL thickness (FIGs. 2D, 2E, upper panels). In contrast, a great majority of the injected locations for the two highest titers (lOxlO ¹¹ and 50xl0 ⁿ vg/mL) showed reduced IS/OS intensity expected from RHO knockdown (FIG. 2D) and some ONL thickening suggesting photoreceptor stress (FIG. 2E). Transition to detectable changes occurred between 5xl0 ^u and 8xl0 ⁿ vg/mL (FIGs. 2D, 2E, arrows) suggesting a range of potentially optimal titer. The great majority of the loci at uninjected sites in the treated eyes at all titers were consistent with results expected from uninjected eyes confirming the localization of the effects of RHO knockdown to the area of the subretinal injection (FIGs. 2D, 2E lower panels).

Animals were humanely euthanized at 7 to 8 weeks post-injection, and four eyes that had been treated with titers ranging from lx to lOxlO ¹¹ vg/mL were processed for histology and rhodopsin immunohistochemistry (FIG. 2F). No obvious qualitative differences in ONL thickness were seen between treated and untreated areas of each eye suggesting that the thickening of the ONL seen by OCT imaging with high viral titers was likely the result of inter or intra cellular swelling (undetectable after tissue fixation) but not cell proliferation. Loss of outer segment structure associated with a prominent reduction of rod opsin immunolabeling was seen in the area treated with the lOxlO ¹¹ vg/mL titer vector. At 5xl0 ⁿ vg/mL some shortening of outer segments and reduction of rod opsin immunolabeling was found in the treated area when compared to the untreated area of the same eye. At the two lowest titers (lx and 2.5xlO ^u vg/mL), outer segments were preserved and rod opsin immunolabeling was comparable between treated and untreated areas. The remaining six eyes injected with titers ranging from lxlO ¹¹ to 50xl0 ⁿ vg/mL (FIG. 2G) were used to assess the efficiency of WY-shRNAs20 in reducing expression of endogenous canine RHO both at the RNA and protein level. Absolute RNA quantification (FIG. 2H) showed very low levels of RHO transcripts (0-3% of that found in untreated areas) in the treated areas of eyes injected with titers ranging from 50xl0 ^u down to 5xl0 ⁿ vg/mL. At lower titers (lxlO ¹¹ to 2.5xlO ⁿ vg/mL) knockdown efficiency was reduced with 22 to 74% of normal RHO RNA levels still remaining in the treated areas. Quantification of RHO protein persisting in the treated areas on immunoblots revealed a dose-dependent effect (FIG. 2H), with undetectable levels in eyes treated with the two highest titers (50xl0 ⁿ and lOxlO ¹¹ vg/mL), 15% in the eye treated with 5xl0 ^u vg/mL, and >47 % with the two lowest titers.

These studies showed that subretinal AAV vector delivery of shRNAsio can achieve very efficient silencing of WT canine RHO, and suggested that the 5xl0 ⁿ vg/mL titer may provide the optimal balance between knockdown of a highly-expressed structural protein in rod photoreceptors without causing major photoreceptor stress or degeneration.

Table 1: Summary of the experimental procedures performed in dogs.

Table 1 ( continued ).

LE: light exposure protocol; OD: right eye; OS: left eye; F: female; M: male; IVit:

intravitreal; subRPE: under the retinal pigment epithelium; cSLO: confocal scanning laser ophthalmoscopy; OCT: optical coherence tomography; RN A/Protein: Quantification of rhodopsin RNA and protein levels; ERG: electroretinography; H&E: histology with hematoxylin & eosin staining; IHC: immunohistochemistry.

Suppression of mutant RHO with shRNAsx

To verify the efficiency of shRNAsio in heterozygous mutant retinas that express both WT and mutant RHO alleles, subretinal injections of AAV-shRNA820 were performed over a range of titers (lxlO ¹¹ to lOxlO ¹¹ vg/mL) in ten ///70-mutant eyes that were followed for 8 to 10 weeks post-injection (Table 1, groups D, E) Since the ///70-mutant dog retinas are highly sensitive to light (45-48), the animals were housed under dim red light from birth until the end of the study, and the surgical intervention was performed under infrared illumination (49). Four eyes were used for quantification of RHO knockdown efficiency at the RNA and protein levels (FIG. 3A; Table 1, group D). As in the WT animals, at a titer of l0xl0 ⁿ vg/mL there was complete silencing of RHO RNA and protein expression in the treated area (FIG. 3B). A similar absence of RHO expression was achieved with the lower (5xl0 ⁿ vg/mL) titer. However, interpretation of this result was confounded by OCT imaging revealing partial loss of ONL thickness restricted to the treated area in this eye. Persistent expression of RHO was seen with the lower (lxlO ¹¹ and 2.5xlO ⁿ vg/mL) titers.

Next, it was evaluated whether knockdown alone could arrest photoreceptor degeneration. Another set of four RHO-mutant eyes (Table 1, group E) were also injected with the same range of titers of ANV-shRNAsx ₎ but at 6 to 8 weeks post-injection they were exposed for one minute to a moderate intensity white light known to cause acute retinal degeneration in this canine model (46-48). Two weeks after light exposure, the eye injected with the titers of lOxlO ¹¹ and 5xl0 ⁿ vg/mL showed a distinct region of ONL retention corresponding to the treatment area (FIG. 3C). Outside the treatment area, there was severe retinal degeneration demonstrating the substantial rescue of photoreceptors achieved by knockdown alone. There were abnormalities with IS/OS intensity expected from knockdown of RHO. Also, the eye injected with the highest (lOxlO ¹¹ vg/mL) titer showed some ONL thickening implying mild photoreceptor stress. Eyes injected with the two lowest titers (2.5xlO ⁿ vg/mL and lxlO ¹¹ vg/mL) had limited to no ONL retention in the treated area (FIG. 3C). Histological analysis of these eyes (FIG. 3D) confirmed the results of in vivo retinal imaging. There was ONL retention with shortened inner segments, loss of outer segment structure, and reduction in rod opsin immunolabeling following injection with the two highest titers. With the 2.5xlO ⁿ vg/mL titer severe ONL thinning was found within the treated area with the exception of a small island of ONL retention. The lowest (lxlO ¹¹ vg/mL) titer did not confer any protection against light exposure. In this eye, the ONL in the treated area resembled that of the untreated region; it was limited to a single row of cone somata with rare residual rod somata and rod opsin-positive debris.

Taken together these findings confirm that shRNAsio can suppress both WT and T4R alleles in vivo, and WY2/5-shRNAs ₂₀ titers in the 5xl0 ^u to lOxlO ¹¹ vg/mL range confer protection of photoreceptor cells (but not their outer segments) from retinal degeneration in RHO- mutant retinas. This partial protective effect likely results from efficient RHO suppression which leads to deconstruction of rod outer segments while keeping the inner segments and rod photoreceptor cell bodies intact. The need to protect the retina from mutant -RHO driven degeneration, while retaining functional rods that have preserved light sensing outer segments, led to exploring whether the suppression of endogenous canine RHO (WT and mutant) could be supplemented with the expression a human RHO cDNA ( RHOsio ) made resistant to shRHAsio-

Combined suppression and replacement

Dual vector - dual function strategy

Initially, a two-vector strategy was tested by co-injecting the AAV-shRNAs2o used above with a similar AAV2/5 serotype carrying the resistant human RHO cDNA (RHOsio) under the control of the human opsin promoter (AAV -RHOsio)· Two RHO-mutant eyes were co-injected with a similar titer (5xl0 ⁿ vg/mL) of both vectors (Treatment 1: 1), and two other mutant eyes were co-injected with APAJ-shRNAsio at 5xl0 ⁿ vg/mL and APAJ-RHOsio at lOxlO ¹¹ vg/mL (Treatment 1:2) (Table 1, group G). One eye from each treatment group was exposed at 7 weeks post-injection to the light exposure protocol, and all four eyes were imaged 4 weeks later by OCT. In the light exposed eye receiving Treatment 1:1, there was some ONL retention, but it did not reach normal thickness in most of the treated area (FIG. 13A). In the region with the greatest ONL retention, there was some rod outer segment preservation suggesting a beneficial outcome conferred by replacement with RHOsio (FIGs. 13B, 13C). Partial ONL protection also occurred in the light exposed eye that had received Treatment 1:2; however, abnormally increased thickness of the inner retina was seen in the treated region (FIG. 13D) resulting from severe perivascular and inner retinal infiltration of mononuclear inflammatory cells (FIG. 13F). In addition, rod outer segment disruption was present (FIG. 13E). Similar findings were observed by OCT in the contralateral shielded eye (Treatment 1:2), that included focal retinal detachment, and signs of perivascular, and subretinal cellular infiltration (FIG. 13G). The results of this two-vector strategy pointed towards a beneficial effect of the combination of knockdown and replacement function.

Nevertheless, there was incomplete rod protection, and treatment resulted in severe retinal complications. To circumvent these limitations, a single AAV vector was developed that combined the knockdown ( shRNAsio ) and resistant replacement (RHOxio) elements. It was hypothesized that this alternative strategy would ensure co-transduction of photoreceptors at a lower viral load, and thus achieve better protection from retinal degeneration and improved safety.

Single vector- dual function strategy

Subretinal injections of ANV-shRNA820-RHOs20 were performed in eight // /70-mutant eyes at the previously determined optimal titer of 5xl0 ^u vg/mL (Table 1, group H). Treated animals were subjected to the light exposure protocol at 7 weeks (n=2 eyes) or at 13 weeks (n=2 eyes) post-injection to determine the efficacy of the single vector approach in preventing acute retinal degeneration. In all four eyes there was substantial retention of ONL thickness 2 weeks after light exposure (FIGs. 4A, 4B; FIGs. 14A, 14B). Most significantly, all four eyes had in the treated area a detectable IS/OS signal. Structural analysis of photoreceptors by IHC (FIGs. 4C, 4D; FIGs. 14C, 14D) confirmed the in vivo results: in treated areas a normal number of photoreceptor cell bodies was retained in the ONL, and rod outer segments were detected. Preservation of elongated rod outer segments was associated with improved morphology of cone inner and outer segments. Four contralateral eyes that had been injected with a similar dose of ANV-shRNA820-RHOs20 but not light exposed were collected at similar time-points (9 and 13 weeks post-injection), and processed for RHO RNA and protein quantification in treated and untreated areas (FIG. 4E). As anticipated, canine RHO RNA (FIG. 4F) at 9 weeks post-injection was considerably reduced in the treated areas (15-16% of that found in untreated areas) of the two eyes. In the two eyes that were processed at 13 weeks post-injection, the levels of remaining canine RHO RNA were further reduced (1-2% of untreated areas). The human RHOs20 transgene transcript levels (FIG. 4G) in the two eyes collected at 9 weeks post-injection were at 5-9% of canine RHO levels measured in untreated areas. At a later time point, 13 weeks post-injection, the levels of human RHOsio RNA were considerably higher (118% to 132% of canine RHO levels measured in untreated areas). At the protein level (FIG. 4G), measurements of total (endogenous canine and human) RHO protein showed a similar temporal trend, with higher RHO protein levels (31-33% of canine RHO levels in untreated areas) at 13 weeks than at 9 weeks post- injection (18-19%). Taken together, these results confirm that a single viral vector that combines both a RHO knockdown and RHO replacement function can effectively preserve the integrity of the entire structure of the rod photoreceptors including their inner and outer segments, and that the levels of expression of the resistant RHO transgene continue to rise several weeks after delivery of the vector.

Thirty-seven week preservation of retinal structure and function with single vector treatment

To assess the long-term stability of the single vector strategy and its ability to protect RHO-matant eyes from degeneration, two ///70-mutant dogs were subretinally injected in one eye with AAN-shRNAsio-RHOsio at the previously determined optimal titer of 5xl0 ⁿ vg/mL, while the contralateral eyes received a similar volume of balanced salt solution (BSS) (Table 1, group I). All four eyes were repeatedly light exposed at 11, 15, 25 and 35 weeks post-injection. OCT imaging was performed pre-injection, as well as immediately prior, and ~2 weeks after each light exposure (FIG. 5A, upper row, timeline). After the first light exposure, there was complete preservation of photoreceptors within the treated area and this dramatic treatment effect persisted for 37 weeks post injection even after three additional light exposures (FIG. 5A, lower rows). Quantitative analysis performed in sampled retinal locations (FIG. 5B, left) from the treated area of the two APAJ-shRNAsio-RHOsio injected eyes showed a small increase in ONL thickness after injection that peaked near 12 weeks before gradually returning to normal levels by 37 weeks (FIG. 5B, middle). IS/OS signal remained detectable at all time-points within the treated areas. There was, a slight decrease in IS/OS intensity that also peaked near 12 weeks followed by a gradual return to normal levels by 37 weeks post injection (FIG. 5B, right).

Electroretinography (ERG) measurements were performed 2.1 - 2.4 weeks after each light exposure to assess retinal function (FIG. 5A, upper row, timeline). Qualitatively, ERGs showed consistently better rod- and cone-mediated function in the AAV -shRNAsio-RHO sio treated eye FIG. 5C, solid line traces) versus the contralateral BSS-injected eye (FIG. 5C, dashed line traces) of a 77770-mutant dog; substantial ERG asymmetry was present between vector and BSS-treated eyes, and the asymmetry increased after each light exposure (FIG. 5C). Quantitatively, amplitudes of rod-dominated ERG traces showed a tendency to decrease over time in both the AAV and BSS injected eyes, likely due to continued photoreceptor degeneration occurring in the peripheral retina outside treatment areas (FIG. 5D, upper panel). Cone function appeared overall to be more stable throughout the 37 week post injection period with four intervening light exposures (FIG. 5D, lower panel). Importantly, at each time point, there were substantially greater rod and cone responses in treated eyes.

These results demonstrate that AAV-shRNAx2o-RHOx2o preserves the integrity of the entire structure of rod photoreceptors, and confers protection of up to 37 weeks of retinal structure and function from the degeneration that otherwise rapidly occurs in untreated RHO~ mutant eyes.

Suppression of canine RHO with Rz525

A hammerhead ribozyme ( Rz525 ) that was shown in an in vitro assay (FIG. 8) to reduce the expression of WT human RHO, was packaged in an AAV2/5 vector and subretinally injected in three WT canine eyes at 20 and 50xl0 ⁿ vg/mL titers (FIG. 9A). The efficiency of AAV2/5 -Rz525 at reducing expression of endogenous canine RHO at the RNA level was measured only in the eye injected with the highest titer (50xl0 ⁿ vg/mL). In the treated area there was complete silencing of RHO expression at the RNA level (FIG. 9B) while RHO protein levels were reduced to -30% (FIG. 9C). The two eyes injected with a 20xl0 ^u vg/ml had higher (47 and 64%) levels of endogenous canine RHO protein remaining in the treated areas (FIG. 9C).

Rz525 was subsequently tested in five heterozygous // /70-mutant eyes (Table 1, group F) that were subretinally-injected with AAV2/5-/¾525 at either 20xl0 ⁿ (3 eyes) or lOOxlO ¹¹ vg/mL (2 eyes). An additional mutant eye was injected with BSS and served as a negative control. At 8 weeks post injection, prominent silencing of RHO expression was seen with the highest titer (lOOxlO ¹¹ vg/mL) both at RNA (13% remaining) and protein (0.1% remaining) levels in the treated area of EM396-OD (FIGs. 10A-10C). However, this treatment was associated with signs of retinal detachment and cellular infiltration in the subretinal space detected by OCT imaging (FIG. 10E, upper row), and confirmed by histology in the fellow eye (EM396-OS) injected with a similar titer (FIG. 10E, lower row). Two eyes injected with the lower 20xl0 ⁿ vg/mL titer had more endogenous canine RHO remaining within the treated area both at the RNA (39 and 66%) and protein (36 and 67%) levels (FIGs. 10A-10C). To evaluate whether these levels of RHO suppression were sufficient to confer protection from light-induced retinal degeneration, two mutant-RHO eyes injected with AAV2/5-/¾525 at 20 and lOOxlO ¹¹ vg/mL were exposed to light at 6 weeks post-injection and imaged by OCT. Two weeks after the light exposure, the eye injected with a titer of lOOxlO ¹¹ vg/mL had some regions of ONL retention within the treated area;

however, no such recue was seen in the eye injected with the lower (20x 10 ¹¹ vg/mL) titer (FIG. 10D).

These results showed that near complete knockdown of RHO could be achieved with Rz525, and that reduction of RHO protein expression was associated with some degree of protection against light- induced retinal degeneration in the canine model of RHO-adRP. Yet, protection could be achieved only when injecting high viral loads that were associated with severe signs of retinitis/chorioretinitis.

Table 2: Codon frequency ( per thousand) at target site of WT RHO and at codon- modified resistant site of hRHO820 (based on Codon Usage Database, kazusa.or.jp/codon/).

Limited suppression of WT canine RHO with shRNAm

A knockdown reagent ( shRNA ) that had been shown to reduce expression of WT human RHO in cell culture (FIGs. 1A-1H) was packaged in an AAV2/5 vector and subretinally injected in two WT canine eyes at 10 and 50xl0 ⁿ vg/mL titers (FIG. 11 A; Table 1, group B). The efficiency of WY2/5-shRNAi _3i at reducing expression of endogenous canine RHO both at the RNA and protein level was limited. Even with the highest titer (50xl0 ⁿ vg/mL) there was still 50% of normal levels of endogenous canine RHO RNA (FIG. 11B) and 70% of endogenous canine RHO protein (FIG. 11C) remaining within the treated areas 8 weeks post-injection. As a result, further investigations in dogs on the use of shRNA as a potential candidate for RHO suppression were not pursued.

Long-term efficacy of AAV 215-RHOs ₂₀ -shRN A ₈₂₀ in preventing the onset of retinal degeneration from repeated light-exposure in canine T4R model of RHO-adRP

The efficacy of the AAV2/5 vector over a period of fifty weeks was evaluated in mutant RHO ^t4r/+ canines. Ability of the vector to confer stable protection of photoreceptors against light- induced retinal degeneration in untreated RHO ^t4r/+ dog retinas was evaluated structurally by OCT and IHC and functionally by ERG. At 12, 24, 36 and 48 weeks post injection, the retinas of these dogs were challenged by an acute light exposure event. OCT and ERG examination were conducted during the course of the study. At termination of the experiment (50 weeks post-injection), retinal tissues were processed for IHC. An illustration of the experimental design is shown in FIG. 17.

ERG measurements showed significantly better rod- and cone-mediated function in the AAV- than in the BSS-treated eyes. FIGs. 18A-18D show representative ERG traces of rod (-1.7 log cd.s.m ² ), mixed rod-cone (0.51 log cd.s.m ² ) recorded dark-adapted, and cone responses to single stimuli (0.51 log cd.s.m ² ) or 29-Hz cone flicker (0.26 log cd.s.m ² ) recorded light-adapted, at ~2 weeks after each light exposure event in a dog injected in one eye with scAA V2/5-hOP-////G,s ₂ -H 1 -.shRNAtoo (dotted line) and with BSS (solid line) in the contralateral eye.

The four RHO- mutant dogs were injected in one eye with scAAV2/5 -RHOsio- shRNAsio (horizontal line shading) and in the contralateral eye with BSS (diagonal line shading) at similar time-points as shown. Longitudinal quantification of maximal amplitudes of rod b-wave, mixed rod-cone a- and b-waves, and of cone lHz and 29Hz flicker responses are displayed in FIGs. 19A-19C. The electrical response from photoreceptors is termed the a- wave and the electrical response from the bipolar cells of the retina is termed the b-wave.

OCT analysis showed preservation of outer nuclear layer (ONL) thickness in the AAV-treated areas while no protection was seen outside of the treated areas nor in the BSS- treated regions. In vivo results were confirmed by histology/IHC that showed preservation of ONL and both rods and cones inner and outer segments. For each of the RHO- mutant dogs, ONL thickness maps and inner segment-outer segment (IS/OS) intensity maps are shown in FIGs. 20A-20D prior to injection (pre-inj.), 12 weeks post- injection (before the first light exposure event), and 50 weeks post-injection (2 weeks after the fourth light exposure event).

A rhodopsin (RHO)/human cone arrestin (hCA) co-immunolabeled retinal cryosection from a RHO-mutant dog illustrates the morphology of the outer nuclear layer (ONL) and outer segments (OS) in untreated and treated areas of the same eye (see FIG. 21). The ONL thickness maps in the lower panel of FIG. 21 shows the approximate location (asterisk) of the retinal untreated and treated areas shown in the upper panel. The transition zone between treated and untreated areas of the eye is shown for reference.

The results show that the A A V2/5-hOP-////(¾ ₂₂ -H 1 -shRNAxio construct confers stable structural protection of photoreceptors against light-induced retinal degeneration up to 50 weeks post-injection in a canine model of RHO-adRP. The results further show that the A A V2/5-hOP-A ^, /7CL' ₂ o-H 1 -shRNAwo construct confers stable functional (electroretinography) protection of photoreceptors against light-induced retinal degeneration up to 50 weeks post injection in a canine model of RHO-adRP. These results show an extension of the above- demonstrated stability of protection by 13 weeks, and confirm structural preservation of rods and cones in the treated area.

Discussion

Despite considerable efforts at developing gene therapies for autosomal dominant diseases(50) only two involving antisense technology (ASO, siRNA) have reached the clinical trial stage, and these are for systemic diseases without a retinal phenotype

(NCT01041222; NCT02363946). The development of mutation-independent gene knockdown and replacement approaches have been explored for the treatment of dominantly inherited systemic and retinal diseases that result from toxic gain-of-function mutations, and/or to circumvent high mutational heterogeneity. (40-43, 51, 52) A significant challenge, that likely has delayed the development of clinical therapies, is the need to successfully fine- tune the level of reduction of both mutant and WT endogenous proteins while providing sufficient resistant replacement. (53) Here, it is shown in a naturally-occurring form of RHO- adRP, and in a large animal model, that this dual-function strategy can effectively provide long-term photoreceptor rescue. In addition, it is shown that when both knockdown and replacement components are co-delivered in the same viral vector, they provide increased efficacy and a better safety profile than when delivered separately. Rapid assessment of gene therapy efficacy in a naturally-occurring large animal model of RHO-adRP

Genetic approaches that include gene augmentation, mutation-dependent RHO suppression, and mutation-independent RHO knockdown and replacement, have been tested to date only in transgenic animal models of RHO-adRP. These include the hP23H mouse (5, 41, 43) the hP347S mouse (36, 38, 42, 54) and the mP23H (lines 1 and 3) rat (22, 23, 25, 26, 33). The use of animal models that have different ratios of mutant transgene to endogenous RHO copy numbers complicates making comparisons of photoreceptor rescue outcomes among these studies, and precludes estimating their potential efficiency in a human RHO- adRP retina. More recently, a P23H opsin knock in mouse that expresses equal levels of murine P23H and WT RHO was generated. (55) However, this model would have had no use in the current study as the target site for shRNA820 in canine and human RHO RNA is not conserved in the mouse.

To increase the predictive value of these studies in the context of a future human clinical trial, the RHO T4R mutant dog was used, which is the only naturally-occurring model of R//(9-adRP(56). Besides its translational value for its human-sized eye, and its phenotypic similarities with Class B patients (56), the RHO ^T4R/+ dog expresses equal amounts of mutant and WT RHO proteins(57). Both forms traffic normally to the rod outer segments(57) and sustain normal retinal structure and function until progressive areas of photoreceptor loss are detected in the inferior-temporal (FIGs. 15A-15C) and central retina (56) within the first two years of life. Sensitivity to light, which has been recognized in other models of RHO-adRP (47) and suspected in Class B patients (19, 45, 58-60), has been well characterized in the canine RHO T4R model. (45) Capitalizing on this light sensitivity, a light exposure protocol was previously developed to experimentally trigger a rapid and synchronized loss of photoreceptors and accelerate the natural disease course. (46) RHO- mutant (but not WT) dogs undergo a complete loss of rods in the central to mid-peripheral retina within two weeks following an acute (1 minute) light exposure using intensity levels encountered in clinical patient settings. (47, 48) Here, this“disease-acceleration” approach was used to obtain a rapid read-out of the effect of gene therapy intervention on preventing rod degeneration in RHO- mutants.

RHO suppression: the need for a potent knockdown component Evidence from several animal model studies suggests that a toxic gain-of-function mechanism is associated with a number of RHO mutations including P23H(55, 61, 62) Tl7M(63, 64) and T4R/T4K(45, 64). This toxicity may be exacerbated following exposure to light in many RHO-adRP models including the /7770-mutant dog (47). Thus, it was posited that under normal ambient illumination, the T4R mutation produces a protein that is highly toxic once bleached, but stable when bound to chromophore (57), and that efficient protection of rods would require significant knockdown of the mutant transcript. This study examined the efficiency of several RHO knockdown reagents including three shRNAs and a

hammerhead ribozyme with the goal of identifying the most potent reagent capable of suppressing RHO expression. Rz525 tested in the 77770-mutant dog produced a 64% reduction in endogenous canine RHO protein that was not sufficient to confer protection from light- induced retinal degeneration (FIGs. 10C-10D). This confirmed the high toxicity of the native mutant T4R protein, since remaining amounts as low as 18% of physiological levels of RHO were sufficient to cause disease in a heterozygous mutant retina, and argued for the need to achieve more efficient suppression. When a complete suppression of RHO protein was obtained with Rz525, some ONL rescue was observed, but the need for a high viral titer (10 ¹³ vg/mL) was associated with severe retinal inflammation (FIGs. 10C-10E). Based on these results, only the most efficient shRNA ( shRNAsio ) identified following in vitro and screening and testing in WT dogs, was subsequently evaluated in mutants. Results confirmed that optimal rescue occurred only when complete suppression of RHO protein expression was achieved, while 86% knockdown of RHO provided only partial protection (FIGs. 3C-3D). Some of the most efficient knockdown reagents reported to date have achieved a 90-95% suppression of human RHO RNA, but these results were obtained on FACS-sorted transduced rods. (36, 41) Here, levels of remaining RHO RNA and protein was intentionally measured from biopsy punches of neuroretina collected within the treated area rather than from an enriched population of transduced rods. The results show a complete knockdown of RHO message and product in 77770-mutant retinas (FIGs. 3B-3C), suggesting not only that shRNAsio is extremely potent but also that rod transduction efficiency was very high.

Importantly, this was achieved with an AAV2/5 titer as low as 5xl0 ^u vg/mF, which previously has been shown to be within the range of well-tolerated titers in dog retinas (44,

65, 66). The near complete suppression of RHO protein expression in WT and 77770-mutant dogs was associated with a loss of OS, similar to the collapse of rod outer segment structure reported by others. (36, 54) It is important to note that suppression of RHO was not associated with any reduction in ONE thickness suggesting that rods can survive for at least 10 weeks following administration of shRNAsx ₎ · This interval provides a window for concomitant expression of a resistant RHO replacement component to produce sufficient protein to prevent outer segment deconstruction or initiate outer segment regeneration.

RHO replacement: how much is enough / too much?

As little as 23 % overexpression of rhodopsin has been shown to cause retinotoxicity in transgenic mice, (67, 68) thus calling for tight regulation of RHO gene supplementation strategies. However, retinal degeneration was not observed when RHO gene augmentation was delivered postnatally in the hP23H RHO ^+/ , mRHO ^+/+ transgenic mouse. This genetic configuration led to a two-fold increase in RHO RNA and a 58% increase in RHO protein, and resulted in both structural and functional rods for up to 6 months post-treatment.(5) These apparently conflicting results suggest that mature rods may tolerate higher levels of RHO overexpression than developing photoreceptors. In the current study, gene

augmentation was not considered in the RHO- mutant dog due to the highly toxic gain-of- function of the T4R mutation, but also because this strategy had failed to confer protection when tested in the hP23H RHO ^+/ , mRHO ^+/ transgenic mouse that carries one mutant (hP23H) and one WT (mRHO) allele. (43) Instead, replacement with a resistant RHO cDNA ( RHO 820 ) was evaluated together with shRNA H2O- mcdi atcd RHO suppression. In the treated areas of mutant retinas injected 9 weeks prior with WY2/5-shRNA ₈₂₀ -RHOs ₂₀ , total RHO protein levels as low as 18% of that found in untreated regions (FIG. 4H) were sufficient to preserve rod outer segment structure (FIG. 4C, FIG. 14C). When retinas from two additional injected eyes were processed 4 weeks later (13 weeks post- injection), higher protein amounts (up to 33% of untreated areas) were measured (FIG. 4H) which also sustained outer segment formation (FIGs. 4D, 14D). These findings suggest that the kinetics of RHO replacement are slower than suppression, and that maximal levels of RHO expression may not be reached until several weeks post treatment.

Combining knockdown and replacement in a single vector achieves optimal efficiency and improved safety over a two-vector approach

Previous efforts at co-packaging the knockdown and replacement reagents within a same viral vector provided short-term (10 days post- injection) preservation of ONL thickness, but failed to rescue rod outer segment structure in a hP23H RHO ^+/ , mRHO ^+/ transgenic mouse.(4l) This led to consideration of a two-vector approach whereby the knockdown and replacement reagents were packaged separately enabling co-administration of different ratios of the two vectors to better control the levels of RHO suppression and replacement. This strategy achieved preservation of ONL thickness, rod outer segment structure, and ERG function in the hP347S RHO ^+/ , mRHO ^+/ transgenic mouse, but the effect was not sustained. (42) In the current study, co-injection of WY -shRNAsio and AA Y-RHOsio led to some degree of protection against light-exposure, yet signs of severe retinal inflammation were observed, likely because of the combined higher viral doses administered (FIGs. 13A-13G). This finding led to the pursuit of the single vector dual-function strategy that was previously successfully evaluated in a mouse model. (43) ShRNAsio , and RHOsio driven respectively by the human Hl RNA and the human opsin proximal promoters were successfully packaged within the cargo capacity limit of the recombinant AAV cassette. The efficiency of this construct remained very high, achieving suppression of ~98.5 % of endogenous canine RHO RNA at 13 weeks post injection, and expression of human RHOsio at levels comparable to normal. Yet, at the protein level, replacement resulted in only about a third of normal levels. This discrepancy between RNA and protein levels could be explained by several factors including the possibility that synonymous codon modifications introduced at wobble/degenerate sites to generate the resistant RHOsio cDNA influenced its translation efficiency. Analysis of codon frequency of the four modified codons at the RHO target site of shRNAsio showed for both dog and man an increase in frequency for two codons, a decrease in frequency for the two others, and an overall decrease when all four were combined (Table 2). Since a correlation has been found between codon usage and relative tRNA abundance in particular for highly expressed genes that are tissue-specific(69) the introduction of two codons with lower frequency could have led to a reduced rate of RHOsio translation. (70) It may be possible, therefore, to improve rhodopsin expression by reducing the number of modifications in the replacement gene. A single mismatch between an siRNA and a mRNA may be sufficient to block RNA silencing, if the mismatch occurs near the RISC-mediated cleavage site. (71) Another possible explanation unrelated to codon bias maybe that the kinetics of RHO suppression are faster than that of RHO replacement. The Hl RNA polymerase III promoter used in the vector is considered safer that the more potent U6 promoter, which leads to a very high level of shRNA expression and, potentially, to saturation of the processing system for endogenous miRNAs. Nevertheless, Hl RNA is expressed abundantly, and the promoter used here functions in all cell types tested. (72, 73) This could explain why at 9 weeks post-injection there was already prominent reduction (~ 84%) of endogenous RHO RNA levels while RHOsio RNA levels reached only 5-9% of normal (FIGs. 4F-4G). While immuno-histochemical analysis revealed the presence of RHO protein in structurally-preserved OS, retinal OCT imaging of the IS/OS intensity, a novel surrogate marker of IS/OS integrity, showed that the signal, although detectable, was decreased at 9 and 13 weeks post-injection (FIGs. 4A-4B, FIGs. 14A-14B). Longitudinal analysis in two ////0- mutant dogs treated with AAN-shRNAsio-RHOsio submitted to four acute light-exposure events showed a similar decline in IS/OS intensity that peaked at ~ 12 weeks post-injection followed by a gradual and near complete recovery by 37 weeks.

Ongoing studies aimed at examining retinal structure at earlier and later time-points after injection will inform as to whether rod outer segment undergo first a deconstruction followed by a gradual reconstruction with disc material composed of resistant RHOxio protein, or whether sufficient levels of RHOxio are produced early enough to prevent outer segment shortening.

Functional assessment

Successful and complete protection of rods was achieved over the long-term (50 weeks / 11.5 months) following a single subretinal injection of APAJ-shRNAsio-RHOsio in mutants that repeatedly had acute light exposures that cause complete loss of rods in the central to mid-peripheral retina after just a single event. Substantially improved ERG responses were consistently seen in AAV-treated eyes at all time points. While cone- mediated ERG response was stable, a slight decline of rod-dominated ERG function was noted. A slight increase in ONL thickness seen in other studies(74, 75) in dogs injected with AAV-mediated gene therapy was also observed here in the treated areas, and likely reflects intra- or intercellular swelling due to mild retinal stress. The transitory and mild ONL thickening was likely associated with the vector since neither the BSS injected eyes before light exposure nor the natural history of uninjected ////0-mutant dogs housed under standard or dim red cyclic illumination demonstrated evidence of abnormal thickening of the ONL (FIGs. 15A-15C). The return of ONL thickness to normal pre-injection values ruled out the hypothesis that the observed ERG decline was associated with photoreceptor loss within the treated regions. Instead, the functional decline is likely explained by the additional loss of rods located in the untreated peripheral retina following cumulative light exposure, and this hypothesis is consistent with the similar decline in rod-dominated ERG amplitudes found in the contralateral BSS-injected eyes.

In summary, a novel single vector with dual RHO knockdown and replacement functions has been developed, that provides complete and long-term protection of rods against a Class B RHO mutation with toxic gain-of-function identified in a naturally- occurring large animal model of RHO-adRP. This highly efficient mutation-independent strategy raises hope that a common gene therapy for all RHO-adRP patients with Class B mutations will be an achievable goal.

Example 2: The construct can protect against retinal degeneration in mice

The ANV-shRNA820-RHO820 construct provided long-term protection against retinal degeneration in a mouse model of RHO-adRP.

C57B1/6 mice transgenic for human P23H RHO are subject to retinal degeneration due to the presence of the mutant rhodopsin gene, even in the presence of dim red lighting without exposure to bright light (33,34,43). At one month of age, mice of this genotype were treated with a subretinal injection of either AAV2/5-GFP or sc A A V 2/5 -RHOs ₂ o-shRNA ₈₂₀ into one eye. The two groups of mice were analyzed at varying intervals: a) pre-treatment, b) 1 month post-injection, c) 2 months post-injection, and d) 3 months post-injection. Subretinal injection induced temporary retinal detachment that ultimately resolved. Contralateral eyes were not treated.

OCT analysis was performed in the P23H RHO mice at monthly intervals for three months to determine the effect of vector treatment on outer nuclear layer (ONL) thickness. ONL thickness was substantially reduced 2 months and 3 months post-injection relative to pre-treatment. In addition, treatment with sc A A V 2/5- RHOs ₂ o-shRN A ₈₂₀ led to a statistically significant preservation of ONL thickness relative to AAV2/5-GFP treatment at all post injection intervals (see FIG. 22).

Overall, thirty P23H mice were successfully treated with scAAV2/5 -RHOs20- shRNAs20 , while twenty-six mice were successfully treated with AAV2/5-GFP. As determined in the Tukey’s multiple comparisons test illustrated in Table 3 below, scAAV2/5- RHO820-shRNAs20 treatment led to a statistically significant degree of protection of retinal structure in P23H RHO mice relative to AAV2/5-GFP treatment.

Dark adapted electroretinography analysis was performed on the same treated groups of P23H RHO mice at monthly intervals for three months. The comeal electrical responses of these mice to brief flashes of light of varying intensities (-20 decibels, -10 decibels and 0 decibels) was measured using comeal electrodes. Longitudinal quantification of maximal amplitudes of rod a- and b- waves (in response to -20 dB and -lOdB flashes) and mixed rod and cone a- and b-waves (in response to 0 dB flashes) at each interval are displayed in FIG. 23. As determined in the student’s t test illustrated in Table 4 below, scAAV2/5 -RHOsio- shRNAsio treatment led to a statistically significant degree of protection of retinal function three months post-injection in P23H RHO mice relative to AAV2/5-GFP treatment (“ns” = not significant).

These results show that the A A V2/5-hOP-/ /7(¾ ₂ o-H 1 -shRNAsio construct confers stable structural protection of photoreceptors against retinal degeneration in a mouse model of RHO-adRP. The results further show that this construct confers stable functional

(electroretinography) protection of photoreceptors against retinal degeneration in this mouse model.

Table 3:

Table 4: p-values

Example 3: Analysis of the mRNA sequences expressed from the vector construct

To determine the types of short mRNA sequences that are derived from the AAV construct, HEK293T cells were transfected with the sc A A V 2/5- RHO.vo-shRN A _H 2 _<> construct. 48 hours later, total RNA was extracted. Extracted RNA was size fractionated and short RNA sequences were subjected to RNA sequencing. RNA molecules with sequences derived from shRNA820were analyzed to determine their sizes and 5’ and 3’ ends (see FIG. 24). The summary of the sequencing reads is shown in FIG. 25. The sequence of the 5’ end of the guide strand (UAG) in 73% of the RNA molecules was equivalent to the expected sequence. 13% of the molecules were shortened by one nucleotide at the 5’ end. 52% had 2 extra uridines at the 3’ end, while 10% had 1 extra uridine at the 3’ end. 13% of molecules had an extra UUA codon at the 3’ end. SEQ ID NOs: 40-46 are provided as the sequences of the most frequent short RNAs derived from shRNAxio (top hits from both strands).

Most of the shRNA was processed as predicted at the 5’ end, with some extra nucleotides present at the 3’ end.

Significance

A number of gene augmentation strategies are entering clinical trials for the treatment of inherited retinal blindness. Gene therapy for autosomal dominant diseases faces significant obstacles that include allelic heterogeneity and the potential need to silence the mutated gene. Here, it is shown that a single gene therapy vector that combines knockdown of the causative gene with its replacement by a resistant wild type copy can prevent photoreceptor cell death and vision loss in canine and mouse models of autosomal dominant retinitis pigmentosa.

Materials and Methods

In vitro assays conducted in HEK293T (ATCC, Manassas, VA) cells (33) were used to screen the efficiency of a hammer-head ribozyme ( Rz525 ) and three short hairpin RNAs ( shRNAm , shRNA U, and shRNAs20 ) at suppressing WT and mutant (P23H, T17M) human RHO expression. (76) Self complementary (77) and non-self complementary AAV vectors were packaged in serotype 5 (78) by triple plasmid DNA transfection and were purified according to previously published methods. (79, 80)The titer of DNase-resistant vector genomes was measured by real-time PCR relative to a standard; purity was validated by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis, sterility and absence of endotoxin were confirmed, and aliquots were stored at -80°C before use. WT and RHO mutant dogs (45, 56) were used to evaluate the response to subretinal injections of AAV2/5 vectors carrying the most potent knockdown reagents, either alone ( Rz525 , shRNA820) or in combination ( shRNA820 ) with a codon-modified resistant human RHO cDNA ( RHO820 ) (FIGs. 16A-16E). Assessment of the effect of RHO suppression and replacement was made by means of enface and cross sectional in vivo retinal imaging, electroretinography, quantification of RHO protein and RNA levels, and morphological evaluation on retinal histological sections. (74, 81-83) A light exposure paradigm (46-48) was used to accelerate the natural course of disease in the RHO mutant dogs and rapidly assess whether the subretinally-delivered AAV constructs prevented the onset of retinal

degeneration. All dogs were bred and maintained at the University of Pennsylvania Retinal Disease Studies Facility (RDSF). The studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and the USDA’s Animal Welfare Act and Animal Welfare Regulations, and complied with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The protocols were approved by an Institutional Animal Care and Use Committee. Methodological details are provided in below.

Ribozyme cleavage assay

HEK293T cells (CRL- 11268, ATCC, Manassas, VA) were transfected with the dual luciferase plasmid psiCHECK™-2 (Promega, Madison, Wi) expressing a 100 nucleotide target region of either wild type or resistant (hardened) human RHO cDNA linked to the Renilla luciferase expressed by the SV40 promoter. RHO transcript levels were measured in six replicates by luciferase assay. The luciferase plasmid was co-transfected with a plasmid expressing Rz525 from the tRNAval promoter. Results were normalized to the same fusion transcript measured following co-transfection with a plasmid lacking ribozyme.

Generation of the R//0-tGFP and RHO ₈₂₀ - tGFP expressing plasmids

A plasmid containing the CMV-promoter, the human WT -RHO open reading frame (ORF) with a C-terminal turboGFP tag, and BGH-PolyA signal (also encoding ampicillin resistance and neomycin resistance genes) was used to express RHO in vitro. P23H RHO, T17M RHO, and RHOsio versions of the CMV-hRHO-turboGFP-BGH-PolyA plasmid were created using the Q5® Site-Directed Mutagenesis Kit (New England Biosciences, Ipswich, MA., USA) according to the manufacturer’s instructions, except with the PCR parameters described here. To generate the P23H RHO version, the 23 ^rd codon of the hRHO ORF of the CMV-hRHO-turboGFP-BGH-PolyA plasmid was changed from CCC to CAC with the following primers: Forward - CACACCCGTCGCATTGGA (SEQ ID NO: 30), and Reverse - GTACGCAGCCACTTCGAGTAC (SEQ ID NO: 31). To produce the RHOsio version, nucleotides 816 to 825 of the hRHO ORF in the CMV-hRHO-turboGFP-BGH-PolyA plasmid were changed from ATTCTACATC (SEQ ID NO: 32) to TTTTTATATA (SEQ ID NO: 33) with the following primers: Forward: ATATATTCACCCACCAGGGCTCCAAC (SEQ ID NO: 34), and Reverse: AAAAAGCCA CGCTGGCGTAGGGC (SEQ ID NO: 35). The PCR reaction parameters were as follows: initial denaturation at 98°C for 30 seconds, 25 cycles of denaturation (98°C for 10 seconds, annealing for 30 seconds, extension at 72°C for 5 minutes), final extension at 72°C for 2 minutes. The annealing temperatures used for the P23H RHO and RHOsio PCR reactions were 68° and 72°C, respectively. 25 pg of the CMV- hRHO-turboGFP-BGH-PolyA plasmid was used as the template for each reaction. The AAV- T17M-GFP was a gift of Dr. Marina Gorbatyuk.(76)

In vitro screening of shRNA-mediated knockdown of RHO

HEK293T cells (ATCC) were seeded in a l2-well plate and transfected the following day when the cells reached 70-90% confluency. Into each well, 500 ng of the CMV-hRHO- turboGFP-BGH-PolyA plasmid expressing either wild-type human RHO, human P23H RHO, human T17M RHO, or RHOsio (a human RHO made resistant to shRNAsio degradation via four silent codon modifications) was transiently co-transfected with lpg of a self

complementary rAAV2 plasmid containing an anti-sense GFP stuffer sequence and either a control Hl-shRNA cassette, an on-target (131, 134, or 820) Hl-shRNA cassette, or no (empty) Hl-shRNA cassette. Each co-transfection condition was performed in triplicate. A DNA to polyethylenimine (PEI at lmg/mL; Polysciences Inc, Warrington, PA., USA) ratio of lpg: 3pL was utilized such that each well received 4.5pL of PEI. The cells were incubated for 48 hours at 37°C with 5% C0 ₂ . Following incubation, the medium was aspirated, and the cells were re-suspended in phosphate buffered saline (PBS) and pelleted by centrifugation at 3,000xg. The PBS was then removed and the cells were re-suspended and sonicated in l50pL of 0.23M sucrose in PBS. 50pL of loading buffer (200 mM Tris-Cl pH 6.8 + 400 mM DTT + 8% SDS + 40% glycerol + bromophenol blue) was applied to each sample and mixed by pipetting. The samples were incubated at room temperature for 30 minutes before being passed through a 28 gauge insulin syringe to shear co-extracted DNA. The total protein concentration of each sample was measured using the Pierce™ 660nm Protein Assay Reagent and the Pierce Ionic Detergent Compatibility Reagent (Thermo Fisher, Waltham, MA., USA). The amount of total protein loaded in to each well (15-20 pg) was constant within each experiment. The samples were run on a 10% Mini-PROTEAN ^® TGX™ Precast Protein Gels (Biorad, Hercules, CA., USA) adjacent to the Li-COR Chameleon ladder (Li-Cor, Lincoln, NE, USA) and transferred to a iBlot PVDF Transfer Stack using Invitrogen’s iBlot system (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Membranes were incubated with methanol for 5 minutes, washed with diH ₂ 0 3 times, and blocked for 1 hour at room temperature with Odyssey blocking buffer (Li-Cor, Lincoln, NE, USA). Membranes were then incubated with mouse anti-TurboGFP (1:2000; Origene, Rockville, MD, USA) and rabbit anti- b -tubulin (1:5000; Millipore, Burlington, MA, USA) in blocking buffer overnight at 4°C, and washed three times with 0.1% Tween20 in PBS before incubation with IRDye 800CW donkey-anti-rabbit and IRDye 680RD goat-anti-mouse (Both 1:5,000; Li-Cor, Lincoln, NE, USA) for 45 minutes at room temperature. Membranes were washed three times with 0.1% Tween20 in PBS and imaged with an Odyssey CLx system (Li-Cor, Lincoln, NE, USA). Band intensity was measured with the ImageJ software. To measure the band intensity of the predicted monomeric form of RHO-GFP, a box was drawn around the prominent band appearing at ~65kDa whereas the aggregated forms were measured using a box drawn from the highest molecular weight marker (260kDa) to the visible band just below 50kDa. Band intensity of RHO was corrected for loading by measuring and dividing by the band intensity of b-tubulin. Values were reported as relative intensity, which was calculated as the corrected band intensity of each sample divided by the average corrected band intensity for the control shRNA condition. Statistical significance was determined via one-way ANOVA followed by Tukey’s multiple comparisons test.

AAV vector preparation.

Adeno-associated virus (AAV) vectors with type 2 terminal repeats (TRs) were packaged in serotype 5 capsids as described by Zolotukhin et al. (2002). (79) AAV5 capsids permit efficient transduction of photoreceptor cells following subretinal injection. (78)

Vectors designed to knockdown human and canine rhodopsin without replacement, contained a 488 bp region (positions 916-1396) of the mouse Rho gene (GenBank M55171.2) and a humanized GFP gene cloned in reverse orientation. This orientation was used to provide a spacer for efficient packaging of AAV without over-expression of GFP. For RHO expression in the RNA replacement vectors, a 536 bp region (positions 4547-5083) from the human RHO gene (GenBank Accession number NG_009l 15.1) was employed as the human rhodopsin proximal promoter (hOP). The promoter was followed by a 163 synthetic intron (SD/SA) from SV40 which preceded 125 base pairs from the RHO 5’ UTR the human RHO cDNA (1046 nt), or a codon-modified version ( RHOsio ) made resistant to shRNAsm (see below). This was followed by a polyadenylation signal from SV40.

The shRNA sense and antisense sequences are shown below, which in each case were connected with the loop sequence UUCAAGAGA (SEQ ID NO: 3).

AAV2/5 vectors for shRNA expression were packaged as self-complementary AAV(77) and expression of shRNAs was directed by the human Hl RNA promoter

(GenBank X16612.1; nucleotides 276-378). The shRNAs contained 19 bp of double stranded sequence connected by a 9 nucleotide loop (UUCAAGAGA, encoded by the sequence TTCAAGAGA). For vectors intended for shRNA delivery without rhodopsin replacement, efficient packaging required maintenance of at least 2.2 kb of DNA between the terminal repeat sequences of AAV. In these vectors, the sequence of humanized GFP (80) was inserted in reverse orientation behind either the mouse opsin proximal promoter or the human opsin proximal promoter (see above). The vector used to express hammerhead ribozyme Rz525, pMOPS500NewHpRz525, used the mouse proximal rhodopsin promoter to drive expression of the ribozyme cassette. It is was described by Gorbatyuk et al. 2007.(33)

Both shRNAsio and a human rhodopsin cDNA ( RHOsio ) made resistant to shRNAsio by introducing silent mutations in the target sequence were packaged together as self complementary AAV2/5. A self-complementary construct was chosen to accelerate the rate of RHO replacement (with RHOsio ) in order to preserve, or rapidly reform rod outer segment structure in the context of a highly efficient KD reagent (shRNAsio).

Animals

All dogs were bred and maintained at the University of Pennsylvania Retinal Disease Studies Facility (RDSF). Studies were carried out in strict accordance with the

recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the USDA’s Animal Welfare Act and Animal Welfare Regulations, and complied with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The protocols were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.

All normal (WT dogs) were housed under standard kennel cyclic (12 hours ON, 12 hours OFF) white light illumination (175-350 lux at the level of the“standard” dog eye). All RHO mutant dogs studied were heterozygous for the T4R mutant allele, and are referred to in text as RHO mutants, and as RHO ^t4r/+ in Table 1 and in the figures to emphasize the heterozygosity. All but 3 mutant RHO dogs were housed under cyclic dim red illumination (9-20 lux at the level of the“standard” dog eye) from birth until termination to prevent any acceleration of retinal degeneration triggered by environmental white light. All

electroretinographic and noninvasive imaging procedures, as well as subretinal injections, were performed under general anesthesia, as previously described. (44, 74, 81) Ocular tissues were collected after euthanasia with i.v. injection of euthanasia solution (Euthasol; Virbac), and all efforts were made to improve animal welfare and minimize discomfort. Included were 21 eyes from 14 normal (WT) dogs, and 40 eyes from 21 mutant RHO dogs (Table 1).

Subretinal injections

Subretinal injections of BSS or vector were performed under direct visualization through an operating microscope (Zeiss OPMI 6; Carl Zeiss Inc, Oberkochen, Germany) and a contact vitrectomy lens using a subretinal cannula as previously reported(65) In the case of the RHO mutant dogs, dim red illumination was set up in the operating room, and subretinal injections were performed under near infrared light using an infrared bandpass filter (RT-830; Hoya Optics, Inc, Fremont, California) placed in the operating miscroscope’s light path and monocular infrared image intensifiers (Owl Nitemare Third generation: BE Meyers & Co,

Inc, Redmond, Washington) that were mounted on the two microscope eyepieces as previously described. (49) This night vision system allows the surgeon to perform subretinal injections in the light-sensitive RHO mutant dogs without causing any surgical light-induced retinal degeneration. (49) Successful subretinal injection of a -150 uL volume produced a bleb that covered -15% of the retinal surface. The location of the subretinal bleb was recorded immediately after each injection. This was done in normal dogs by fundus photography (Retcam shuttle, Clarity Medical Systems, Pleasanton, CA), and in // /0-mutant dogs by drawings of the bleb on near infrared cSLO composite images captured prior to the injection.

Experimental acceleration of retinal disease by a light exposure.

An acute light exposure protocol was used as previously described (46-48) to assess the efficiency of the viral vector constructs at preventing retinal degeneration in the light sensitive ////0- mutant dog. (45) Ah steps of this procedure were carried out under dim red light illumination. The pupils were dilated with 1% tropicamide and 1% phenylephrine (3 times, 30 minutes apart in both eyes), and general anesthesia was induced with propofol (4mg/kg) IV and maintained with inhalation anesthesia (isoflurane). To prevent the ventral rotation of the globe induced by the general anesthesia, a retrobulbar saline injection (5-10 ml) was performed to recenter the eyes in the primary gaze position. A one minute exposure to white (6500K) light at a comeal irradiance of 1 mW/cm ² (measured with a luminometer, IL1700; International Light Technologies, Peabody, MA, USA) was performed using a monocular Ganzfeld stimulator (ColorBurst; Diagnosys LLC, Lowell, MA, USA) from an ERG system (Espion, Diagnosys LLC). Eyes that were not exposed were kept shielded with a black photographic cloth during the light exposure procedure in the contralateral eye.

In vivo optical coherence tomography (OCT) imaging and analyses

Enface and retinal cross-sectional imaging was performed with the dogs under general inhalation anesthesia as described above. Overlapping enface images of reflectivity with near-infrared illumination (820 nm) were obtained (Spectralis HRA+OCT, Heidelberg, Germany) with 30° and 55° diameter lenses to delineate fundus features such as optic nerve, retinal blood vessels, boundaries of injection blebs, retinotomy sites and other local changes. Custom programs (MatLab 7.5; The MathWorks, Natick, MA) were used to digitally stitch individual photos into a retina-wide panorama. Two methods were used to overlay injection blebs onto panoramic images. In WT eyes, photos of blebs taken at the time of the surgery were registered and bleb boundaries were transferred. In RHO- mutant eyes, sketches of blebs drawn at the time of the surgery were transferred onto panoramic images.

Spectral-domain optical coherence tomography (SD-OCT) was performed with linear and raster scans (Spectralis HRA+OCT, Heidelberg, Germany). Overlapping (30°x20°) raster scans were recorded covering large regions of the retina. Post-acquisition processing of OCT data was performed with custom programs (MatLab 7.5; The MathWorks, Natick, MA). For retina-wide topographic analysis, integrated backscatter intensity of each raster scan was used to locate its precise location and orientation relative to retinal features visible on the retina wide panorama. Individual longitudinal reflectivity profiles (LRPs) forming all registered raster scans were allotted to regularly spaced bins (l°xl°) in a rectangular coordinate system centered at the optic nerve; LRPs in each bin were aligned and averaged. Intensity and slope information of the backscatter signal along each LRP was manually evaluated to segment two boundaries that define the ONL. One boundary was the distal transition of the outer plexiform layer (OPL) peak. The other boundary was the external limiting membrane (ELM) peak. In locations with severe retinal degeneration without a detectable ELM peak, the second ONL boundary was placed at the most proximal transition to the RPE peak. In addition, the normalized IS/OS backscatter intensity was calculated by subtracting the mean backscatter intensity of the 5 axial samples vitreal to the OPL boundary from the mean backscatter intensity of the 5 axial samples scleral to the ELM boundary; the latter included the IS/OS peak.(44, 74) IS/OS intensities were only mapped in regions of retained inner and outer segment length since compromise of the latter made it impossible to distinguish the IS/OS signal from the RPE/tapetum signal. Topographic results from uninjected control eyes were registered by the center of the ONH and the canine fovea(83) and maps of control variability were generated defining the 99 ^th percentile confidence intervals. Injected eyes were compared locus-by-locus to the control confidence intervals to generate maps of significant change.

Electroretinography (ERG) recording and analyses

Dogs were pre-medicated with subcutaneous injections of atropine, and

acepromazine, and their pupils dilated with atropine (1%), tropicamide (1%) and phenylephrine (10%). After induction with intravenous propofol, dogs were maintained under general inhalation anesthesia (isoflurane), and their pulse rate, oxygen saturation and temperature was monitored for constancy during the entire procedure. Full-field flash electroretinography was performed as previously described(44, 74) on both eyes using a custom-built Ganzfeld dome fitted with the LED stimuli of a ColorDome stimulator

(Diagnosys LLC, Lowell, MA). After 20 minutes of dark adaptation, rod and mixed rod- cone-mediated responses (averaged 4 times) to single 4 ms white flash stimuli of increasing intensities (from -3.74 to 0.51 log cd.s.m ² ) were recorded. Following 5 minutes of white light adaptation (1.53 log cd.m ² ), cone-mediated signals (averaged 10 times) to a series of single flashes (from -2.74 to 0.51 log cd.s.m ² ) and to a 29.4-Hz flicker (averaged 20 times; from - 2.74 to 0.26 log cd.s.m ² ) stimuli were recorded. Waveforms were processed with a digital low-pass (50 Hz) filter to reduce recording noise if necessary. Amplitudes of the a- and b- waves of the scotopic mixed rod-cone ERG, and the peak to peak amplitudes of the photopic single flash and 29.4 Hz cone flicker were measured.

Retinal histology and immunohistochemistry

Following euthanasia and enucleation, the globes were fixed in 4% paraformaldehyde (PFA) for 3 hours followed by 2% PFA for 24 hours, trimmed, cryoprotected in 15-30% sucrose/PBS solution, and embedded in optimal cutting temperature media as previously reported(81). Ten-micro meter- thick serial sections that encompassed the nontreated, the boundary, and the treated/bleb area were cut on a cryostat (Microm HM550; Thermo Fisher Scientific, Kalamazoo, MI). Blood vessel landmarks identified by H&E staining were used to determine the precise location of the retinal cryosections on the vascular pattern of the en face cSLO images, as previously reported. (44, 74, 81) Sequential sections were

immunolabeled with primary antibodies and cell-specific markers: rod opsin (cat#

MAB5316; 1:200 dilution; EMD Millipore, Billerica, MA), goat anti-human cone arrestin (W. Beltran, Univ. of Pennsylvania; 1:100). The antigen-antibody complexes were visualized with fluorochrome-labeled secondary antibodies (Alexa Fluor, 1:200; Molecular Probes, Kalamazoo, MI), and Hoechst 33342 nuclear stain (Molecular Probes) was used to label cell nuclei. H&E-stained sections were examined by widefield microscopy (Axioplan; Carl Zeiss Meditec, Dublin, CA), and the images were digitally captured (Spot 4.0 camera; Diagnostic Instruments, Sterling Heights, MI) and imported into a graphics program (Illustrator; Adobe, San Jose, CA) for display. Sections labeled for fluorescent immunohistochemistry were examined by confocal microscopy (Leica TCS SP5; Leica Microsystems, Buffalo Grove, IL), and digital images were taken, processed using the Leica Application suite program, and imported into a graphics program (Illustrator; Adobe).

Retinal tissue sampling for RHO RNA and protein quantification

Immediately following enucleation and separation of the posterior cup, 3 mm biopsy punches from treated and untreated neuroretinal areas were individually collected in cryovials, frozen in liquid nitrogen and stored at - 80° C. For RHO mutant dogs retinal sampling was performed under dim red illumination.

RNA extraction and cDNA synthesis

Total RNA was extracted from the punches of neuroretina using Direct- zol RNA Miniprep Kit (Zymo Research, Irvine, CA). cDNA was prepared from total RNA using the High Capacity RNA to cDNA kit (Applied Biosystems, Foster City, CA) following the manufacturer’s recommendations.

Absolute quantification of canine and human RHO transcripts in retina

To efficiently determine the ratio between endogenous canine and exogenous human RHO transcripts in the same retinal samples specific primer pairs have been designed for canine (For: 5’ -AC AAGACGGGTGTGGTGCGC (SEQ ID NO: 17); Rev: 5’- TCATGGGCGTCGCCTTCACC (SEQ ID NO: 18)) and human RHO (For: 5’- CCATCAACTTCCTCACGCTCTA (SEQ ID NO: 19); Rev: 5’-

TAGGTTGAGCAGGATGTAGTTGAGA (SEQ ID NO: 20)). The SYBR green platform was used for the analysis using a primer concentration of 0.15 mM. Real-time PCR was performed in a total volume of 25 pL in 96-well microwell plates on the Applied Biosystems 7500 Real-Time PCR System. All PCRs were performed using cDNA generated from 0.1 ng DNAase-treated RNA. The RT-PCR product was used for construction of an absolute standard curve for individual amplicons representing the canine and human RHO. The number of copies of a template was calculated as previously described. (82) The dynamic range of the calibration curves was between 10 ³ and 10 ⁷ molecules. Amplification data were analyzed with the 7500 Software version 2.0.1 (Applied Biosystems).

Quantification of Rhodopsin protein:

Protein retinal extracts were prepared from 3 mm biopsy punches collected (under dim red illumination for RHO mutant dogs) from treated and untreated neuroretinal areas. After sonication in a buffer containing 0.23M Sucrose, 2 mM EDTA, 5mM TrisHCl, pH 7.4, and protease inhibitors (Halt Protease Inhibitor cocktail, cat. No. 87786, Thermo Fisher Scientific, Waltham, MA.), samples were centrifuged and total protein concentration in the supernatant was measured by the Bradford method. One mg of total protein from each sample was resolved on 8-16% Tris Glycine gel (Invitrogen, Carlsbad, CA), transferred to a nitrocellulose membrane (iBLOT, Invitrogen) and immunoblotted using anti-Rhodopsin antibody (MAB5316, 1:1000 dilution, EMD Millipore, Billerica, MA) and anti-Histone H3 antibody (abl79l, 1:3000 dilution, Abeam, Cambridge, MA). Protein bands were visualized on a digital imaging system (Odyssey Fc, Licor, Lincoln, NE) after incubation with infrared labeled secondary antibodies (IRDye 680 and IRDye 800, Licor). Amounts of Rhodopsin protein were quantified with the Licor Image Studio v4.0 software using the histone H3 band for normalization.

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OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined in any

combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles“a” and“an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean“at least one.”

The phrase“and/or,” as used herein in the specification and in the claims, should be understood to mean“either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e.,“one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to“A and/or B”, when used in conjunction with open-ended language such as“comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims,“or” should be understood to have the same meaning as“and/or” as defined above. For example, when separating items in a list,“or” or“and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as“only one of’ or“exactly one of,” or, when used in the claims,“consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term“or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e.“one or the other but not both”) when preceded by terms of exclusivity, such as“either,”“one of,”“only one of,” or “exactly one of.”“Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase“at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase“at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example,“at least one of A and B” (or, equivalently,“at least one of A or B,” or, equivalently“at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another

embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one,

B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,”“including,”“carrying,”“having,� �“containing,”“involving,”“holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases“consisting of’ and“consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g.,“comprising”) are also contemplated, in alternative embodiments, as“consisting of’ and“consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the disclosure describes“a composition comprising A and B”, the disclosure also contemplates the alternative embodiments“a composition consisting of A and B” and“a composition consisting essentially of A and B”.