Gene therapy vector

Abstract

The present invention relates to a polynucleotide comprising two inverted terminal repeats (ITRs), an hGRK1 promoter, an SV40 late intron, a transgene encoding an RPGR polypeptide, and an SV40 late polyA.

Description

[0001] believed that the following factors contribute to the safety and efficacy of the polynucleotides, viral particles and compositions of the invention:

[0002] (1) the use of a transgene encoding a full-length RPGR ORF 15 polypeptide;

[0003] (2) the use of the particular hGRKl promoter;

[0004] (3) the use of an AAV2-derived capsid; and / or

[0005] (4) a high full: empty ratio of the viral particles.

[0006] Accordingly, in a first aspect, the present invention provides a polynucleotide comprising two inverted terminal repeats (ITRs), an hGRKl promoter, an SV40 late intron, a transgene encoding an RPGR polypeptide, and an SV40 late polyA.

[0007] In a second aspect, the present invention provides a viral particle comprising a recombinant genome comprising the polynucleotide of any of the preceding embodiments and a capsid.

[0008] In a third aspect, the present invention provides a composition comprising a multiplicity of the viral particles of the invention.

[0009] In a fourth aspect, the present invention provides a method for producing a composition comprising a multiplicity of AAV particles, wherein the method comprises a step of performing anion exchange chromatography on a sample comprising the AAV particles.

[0010] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the polynucleotide, the viral particle, or the composition of the invention.

[0011] In a sixth aspect, the present invention provides the polynucleotide, the viral particle, the composition, or the pharmaceutical composition of the invention for use in a method of treatment in a subject, wherein the method comprises administering the polynucleotide, the viral particle, the composition or the pharmaceutical composition to the eye.

[0012] In a seventh aspect, the present invention provides a method of treatment in a subject, the method comprising administering the polynucleotide, the viral particle, the composition, or the pharmaceutical composition of the invention to the eye. In an eight aspect, the present invention provides the polynucleotide, the viral particle, the composition, or the pharmaceutical composition of the invention for use in the manufacture of a medicament for use in a method of treatment.

[0013] Description of the Figures

[0014] Figure 1 Schematic of hGRKl-RPGR vector genome

[0015] Fig. 1 shows a schematic of the hGRKl-RPGR vector genome comprising, from 5’ to 3’, an AAV2 ITR, hGRKl promoter, SV40 late intron, codon-optimised RPGR transgene encoding ORF 15, SV40 polyA tail, and a second AAV2 ITR.

[0016] Figure 2 Retinal sensitivity (MAIA microperimetry)

[0017] Fig. 2 shows the number of responders (defined as a patient achieving a 7dB or greater improvement from baseline in at least 5 loci) in the study and fellow eyes of the low dose (7.5 x 1010vg / eye) and high dose (6.8 x 1011vg / eye) groups as measured by MAIA microperimetry at Months 12 and 24. (A) While no low-dosed eyes were responsive at Month 12, 5 / 8 (63%) high dose-treated eyes showed response (none of the corresponding fellow (untreated) eyes were responders). (B) A consistent effect was observed at Month 24, although subject to skewing from loss-to-follow-up of one high-dose and two low-dose patients. 25% and 57% of low- and high-dosed eyes were responders at Month 24, with no fellow eyes showing response.

[0018] Figure 3 Retinal sensitivity (MAIA microperimetry)

[0019] Fig. 3 shows the mean change from baseline (CFB) in mean sensitivity (as measured by MAIA microperimetry) + / - standard error across all patients. Mean changes from baseline over time in mean retinal sensitivity for loci within the whole grid showed a clear dose response in favour of the high dose group. SE = study eye; FE = fellow eye.

[0020] Figure 4 Example of responding eye as measured by MAIA microperimetry

[0021] Fig. 4 is a composite example of a responder participant in the high dose group as demonstrated by MAIA microperimetry heat maps and retinal sensitivity change by plotting the individual loci within the whole grid. Fig. 4A shows the heat maps for the treated and untreated eyes. Positive visual sensitivity change is evident in the treated eye, increasing in both magnitude of effect as well as the overall area of effect from baseline to Month 12. Fig.

[0022] 4B also shows that there are at least 5 loci at Month 12 and, again, at Month 24 with a 7 dB or greater improvement in the study. A marked difference is also seen between the treated and untreated eye in this participant.

[0023] Figure 5 Mean Change from Baseline in Mobility Maze Score

[0024] Ora- Visual Navigation Challenge (VNC™) assessment demonstrated positive trends in the high dose group, with 5 / 8 treated eyes (study eyes; SE, connected by solid lines) available for assessment showing at least a one level improvement in the VNC™, relative to 0 / 8 of the available untreated eyes (fellow eyes; FE, connected by dashed lines) at Month 12 (Fig. 5A), and 5 / 7 study eyes available for assessment showing at least a one level improvement in the VNC™, relative to 1 / 6 of the available fellow eyes at Month 24 (Fig. 5B). For each paired data point, the study eye is shown on the left and the fellow eye on the right. No Month 12 data were available for the shaded data points. LCVNC = low contrast visual navigation challenge; HCVNC = high contrast visual navigation challenge.

[0025] Figure 6 Mean Change from Baseline in LLVA

[0026] Fig. 6 shows mean change from baseline (CFB) LLVA data through to Month 3 for Group 1 and 2 study eyes, and the fellow eyes (combined Groups 1 and 2). While the fellow (i.e. previously -treated) eyes remained stable over the period, significant increases were observed for both groups of study (i.e. currently-treated) eyes.

[0027] Figure 7 Change from Baseline in LLVA by subject

[0028] Fig. 7 presents Month 3 results for the proportion of study and fellow eyes falling into LLVA response categories (<10 ETDRS letters; 10-14 letters; > 15 letters), respectively plotted against baseline low luminance deficit (LLD) (Fig.7A), baseline BCVA (Fig.7B) and baseline LLVA (Fig.7C). The study eye data in each case are ordered as waterfall, with the fellow eye data ordered on a corresponding subject basis as indicated by the notation at the top of each bar (HD = high dose; LD = low dose). LLD is the difference between the BCVA and LLVA values. Figure 8 Mean Change from Baseline in LLVA

[0029] Fig. 8 shows mean change from baseline (CFB) LLVA data through to Month 9 for Group 1 and 2 study eyes, and the fellow eyes (combined Groups 1 and 2). While the fellow (i.e. previously -treated) eyes showed only minor improvement over the period, significant increases were observed for both groups of study eyes, in particular reaching an average increase of 16 letters by month 9. (HD = high dose; LD = low dose; FE = fellow eye; SE = study eye)

[0030] Figure 9 Change from Baseline in LLVA by subject

[0031] Fig. 9 presents Month 6 to Month 12 (based on the latest study visit for each patient) results for the proportion of study and fellow eyes falling into LLVA response categories (<10 ETDRS letters; 10-14 letters; > 15 letters), respectively plotted against baseline low luminance deficit (LLD). The study eye data in each case are ordered as waterfall (i.e. from highest LLD to lowest LLD), with the fellow eye data ordered on a corresponding patient basis as indicated by the notation at the top of each bar. LLD is the difference between the BCVA and LLVA values. An apparent correlation between high baseline LLD and high LLVA response is evident from Fig. 9. (HD = high dose; LD = low dose; M6 = Month 6; M9 = Month 9; M12 = Month 12)

[0032] Figure 10 Change from Baseline in LLVA by subject

[0033] Fig. 10 presents Month 6 to Month 12 (based on the latest study visit for each patient) individual LLVA results for each study and corresponding fellow eye. The data shows that 57% of the study eyes in the high dose group show a >15 letter increase in LLVA score, while 86% show an increase of at least 10 letters. 100% of the low dose group showed at increase of at least 10 letters. (HD = high dose; LD = low dose; M6 = Month 6; M9 = Month 9; M12 = Month 12).

[0034] Figure 11 Example of responding eye as measured by MAIA microperimetry

[0035] Fig. 11 is a composite example of a responder participant in the high dose group as demonstrated by MAIA microperimetry heat maps and retinal sensitivity change by plotting the individual loci within the whole grid. The arrow timeline refers to time (T) in months. Fig. 11 A shows the heat maps for Eye 1 (treated first; Example 1 study) and Eye 2 (treated second; Example 4 study). The time points for each eye show the number of months after treatment for each eye. Positive visual sensitivity change is evident in both treated eyes, increasing in both magnitude of effect as well as the overall area of effect from baseline to Month 24 (Eye 1) and from baseline to Month 12 (Eye 2). Fig. 11B also shows that there are at least 5 loci at Month 12 and, again, at Month 24 with a 7 dB or greater improvement in the study in Eye 1 relative to baseline. In Fig. 11B, Treated refers to Eye 1, while Untreated refers to Eye 2 (baseline was taken at the same time for both eyes). Fig. 11C shows that there are at least 5 loci at Months 6, 9 and 12 with a 7 dB or greater improvement in the study in Eye 2 relative to baseline. In Fig. 11C, Treated refers to Eye 2, while Untreated refers to Eye 1 (baseline was taken at the same time for both eyes).

[0036] Detailed Description

[0037] General definitions

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by a person skilled in the art to which this invention belongs.

[0039] In general, the term “ comprising" is intended to mean including but not limited to. For example, the phrase “a viral particle comprising a capsid" should be interpreted to mean that the viral particle has a capsid, but the viral particle may comprise further elements. In some embodiments of the invention, the word "comprising ' may be replaced with the phrase “consisting of. The term “consisting of is intended to be limiting. For example, the phrase “a viral particle consisting of a capsid and a recombinant genome" should be interpreted to mean that the viral particle has a capsid and a recombinant genome and contains no further components.

[0040] In some embodiments of the invention, the word “comprising” may be replaced with the phrase “consisting essentially of’. The term “consisting essentially of” means that specific further components can be present, namely those not materially affecting the essential characteristics of the subject matter. The singular forms “a”, “an”, and “the” include plural referents unless the context clear dictates otherwise. Thus, for example, reference to “an amino acid” includes one or more instances or versions of such amino acids.

[0041] The terms “protein” and “polypeptide” are used interchangeably herein, and are intended to refer to a polymeric chain of amino acids of any length.

[0042] The term “fragment” as used herein refers to a contiguous portion of a reference sequence. For example, a fragment of an AAV capsid may refer to at least 100, at least 200, at least 300, at least 400, or at least 500 contiguous amino acids of the AAV capsid.

[0043] The terms “nucleic acid molecule”, “polynucleotide” and “nucleotide sequence” are intended to refer to a polymeric chain of any length of nucleotides, including deoxyribonucleotides, ribonucleotides, or analogues thereof. For example, the nucleic acid molecule, polynucleotide or nucleotide sequence may comprise DNA (deoxyribonucleotides) or RNA (ribonucleotides). The nucleic acid molecule, polynucleotide or nucleotide sequence may consist of DNA. The nucleic acid molecule, polynucleotide or nucleotide sequence may be mRNA. Since the nucleic acid molecule, polynucleotide or nucleotide sequence may comprise RNA or DNA, all references to T (thymine) nucleotides may be replaced with U (uracil).

[0044] For the purpose of this invention, in order to determine the percent identity of two sequences (such as two polynucleotide or two polypeptide sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in a first sequence for optimal alignment with a second sequence). The nucleotide or amino acid residues at each position are then compared. When a position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence, then the amino acids are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions in the reference sequence x 100).

[0045] Typically, the sequence comparison is carried out over the length of the reference sequence. For example, if the user wished to determine whether a given (“test”) sequence has at least 80% identity to SEQ ID NO: 1, SEQ ID NO: 1 would be the reference sequence. To assess whether a sequence has at least 80% identity to SEQ ID NO: 1 (an example of a reference sequence), the skilled person would carry out an alignment over the length of SEQ ID NO: 1, and identify how many positions in the test sequence were identical to those of SEQ ID NO: 1. If at least 80% of the positions are identical, the test sequence is at least 80% identical to SEQ ID NO: 1. If the sequence is shorter than SEQ ID NO: 1, the gaps or missing positions should be considered to be non-identical positions.

[0046] To assess whether a sequence is at least 80% identical to a fragment of 450 amino acids of SEQ ID NO: 9, the skilled person would align SEQ ID NO: 9 to the test sequence, and determine which contiguous 450 amino acids of SEQ ID NO: 9 best align to the test sequence. The skilled person would then determine the number of positions in the test sequence that are identical to the 450 amino acids of SEQ ID NO: 9 which best align to the test sequence, and calculate the percentage identity as indicated above.

[0047] The skilled person is aware of different computer programs that are available to perform an alignment between two sequences. An alignment between two sequences can be accomplished using a mathematical algorithm. For example, an alignment may be performed using the Needleman and Wunsch algorithm (Needleman and Wunsch, 1970, J Mol Biol.;48(3):443- 53) which aligns the sequences optimally over the entire length). Sequences of substantially different lengths may alternatively be aligned using a local alignment algorithm (e.g.15 Smith and Waterman algorithm (Smith and Waterman, 1981, J Theor Biol.;91 (2):379-80) or Altschul algorithm (Altschul SF et al., 1997, Nucleic Acids Res.;25(17):3389-402,; Altschul SF et al., 2005, Bioinformatics.;21(8): 1451-6). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0048] In some cases, a polypeptide or polynucleotide of the invention may be identical to a reference sequence, “but for” one or more substitutions or insertions. As used herein, “identical to SEQ ID NO: X, but for a substitution” means that the polypeptide or polynucleotide has the same sequence as SEQ ID NO: X, other than the specified substitution(s). For example, a polypeptide that is identical to SEQ ID NO: 10, but for a T to G substitution at position 199 is a sequence that is otherwise identical to SEQ ID NO: 10, except at position 199, where the polypeptide comprises a G in place of an T. In order to determine whether a polynucleotide is identical to a reference sequence but for a specific substitution or insertion, the person skilled in the art merely needs to perform a sequence alignment of the nucleotide sequence of the polynucleotide with the specified SEQ ID NO using a suitable alignment algorithm such as that of Needleman and Wunsch described above, and determine whether the sequence is the same at all positions other than the specified position. For example, the skilled person is able to align the amino acid sequence of a polypeptide with SEQ ID NO: 10 and determine whether it is identical to SEQ ID NO: 10, but for a T to G substitution at position 199.

[0049] A polypeptide or polynucleotide may comprise an insertion between two amino acids or two nucleotides and in these cases the inserted sequence is added relative to a reference sequence. For example, a polynucleotide sequence may be identical to SEQ ID NO: 10 but for a GCC insertion between positions 246 and 247 if the sequence GG at positions 246 and 247 is replaced with GGCCG.

[0050] The terms “wild-type” and “native” are used interchangeably herein, and are intended to describe something which is naturally occurring. For example, a “native RPGR polypeptide” is an RPGR polypeptide which occurs in nature such as an RPGR polypeptide of SEQ ID NO: 11. A wild-type or native RPGR transgene is an RPGR sequence which occurs in nature such as an RPGR transgene of SEQ ID NO: 19.

[0051] The terms “subject” and “patient” are used interchangeably herein and refer to an individual to whom a treatment is administered.

[0052] Throughout the application the mathematical terms “XE+Y” and “Xx 10' are used interchangeably. For example, 6.8E+11 is equal to 6.8 x 1011.

[0053] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Transgene

[0054] The polynucleotides and viral particles of the invention comprise a transgene. The term "transgene" refers to a nucleic acid sequence (typically encoding a protein) comprising a gene of interest (commonly a heterologous gene). The transgene is generally not an AAV-derived sequence, and may for example encode a human protein or a protein from another organism. The transgene is typically, but not necessarily, of the same origin as the subject to be treated with the viral particle. The term “a transgene" should be construed as comprising one or more transgenes.

[0055] In the present invention, the transgene encodes an RPGR polypeptide. In some embodiments, the transgene comprises a nucleotide sequence at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 2. In some embodiments, the transgene comprises a nucleotide sequence at least 99.5% identical to SEQ ID NO: 2. In some embodiments, the transgene comprises a nucleotide sequence identical to SEQ ID NO: 2. In some embodiments, the transgene comprises a TAA stop codon. Optionally, the transgene comprises a nucleotide sequence at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 2, wherein the nucleotide sequence comprises a T at a position corresponding to position 3457, an A at a position corresponding to position 3458 and an A at a position corresponding to position 3459. A nucleotide will be considered to correspond to a nucleotide at position 3457, 3458 or 3459 if it aligns to position 3457, 3458 or 3459. Optionally, the transgene comprises a TGA stop codon, but preferably the transgene does not comprise a TGA stop codon. Optionally, the stop codon corresponds to nucleotides 3457, 3458 and 3459 of SEQ ID NO: 2. In some embodiments, the transgene comprises a nucleotide sequence at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 18. In some embodiments, the transgene comprises a nucleotide sequence at least 99.5% identical to SEQ ID NO: 18. In some embodiments, the transgene comprises a nucleotide sequence identical to SEQ ID NO: 18.

[0056] In some embodiments, the transgene comprises a nucleotide sequence at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 15 or 16. In some

[0057] 10 embodiments, the transgene comprises a nucleotide sequence at least 99.5% identical to SEQ ID NO: 15 or 16. In some embodiments, the transgene comprises a nucleotide sequence identical to SEQ ID NO: 15 or 16.

[0058] In some embodiments, the transgene comprises a nucleotide sequence that is identical to SEQ ID NO: 7 but for one or more substitutions selected from a substitution to T at position 30; C at position 33; C at position 49; G at position 57; C at position 60; A at position 69; G at position 71; T at position 170; T at position 189; C at position 441; C at position 537; G at position 546; C at position 786; C at position 792; G at position 966; G at position 969; C at position 990; C at position 1011; C at position 1014; C at position 1029; G at position 1299; G at position 1689; C at position 3355; A at position 3357; A at position 3363; A at position 3403; G at position 3404; C at position 3405; G at position 3408; T at position 3409; C at position 3410; C at position 3432; A at position 3438; and G at position 3456.

[0059] The transgene may comprise a nucleotide sequence that is identical to SEQ ID NO: 7, but for 2 or more, 3 or more, 4 or more, 5 or more, 10, or more, 15 or more, or 20 or more of the substitutions recited in the previous paragraph. In some embodiments, the transgene comprises 15 or more of the substitutions recited in the previous paragraph. In some embodiments, the transgene comprises all of the substitutions recited in the previous paragraph.

[0060] In some embodiments, the transgene is codon optimised. A nucleotide sequence can be codon optimised by replacing codons with other codons that are favoured (i.e. reflective of codon bias) in a particular organ or a particular organism (so-called favoured codons). Such a codon optimisation improves expression of the nucleotide sequence in the particular organ or particular organism. For example, if a nucleotide sequence is codon optimised for the human eye, the nucleotide sequence is modified to increase the number of codons that are favoured in the human eye. To identify whether an RPGR transgene is codon optimised, the skilled person can align the RPGR transgene with a wild-type RPGR transgene (for example SEQ ID NO: 19). The skilled person can then determine whether the sequence is codon optimised, z.e., whether it comprises at least one codon that has been replaced with a favoured codon. In some embodiments, the favoured codons for the RPGR transgene are GCC, TGC, GAC, GAG, TTC, GGC, CAC, ATC, AAG, CTG, AAC, CCA, CCC, CAG, CGG, CGA, CGC, AGT, AGC, TCC, ACC, GTG, GTC, and TAC. In some embodiments, the RPGR transgene comprises more favoured codons than the RPGR transgene of SEQ ID NO: 19.

[0061] In some cases, not all of the codons encoding a particular amino acid will be changed to the favoured codon. For example, expression may be optimised by converting only a specific portion (e.g. 50%) of codons encoding a specific amino acid to favoured codons.

[0062] In some embodiments, at least 75% of the codons encoding alanine residues in the transgene are GCA or GCC. In some embodiments, the remaining codons encoding alanine are GCT, i.e. none of the codons encoding alanine residues are GCG. In some embodiments, at least 40% of the codons encoding alanine residues in the transgene are GCC, optionally about 43%.

[0063] In some embodiments, at least 50% of the codons encoding cysteine residues in the transgene are TGC, optionally wherein about 60% of the codons encoding cysteine residues in the transgene are TGC.

[0064] In some embodiments, at least 30% of the codons encoding aspartate residues in the transgene are GAC, optionally wherein about 50% of the codons encoding aspartate residues in the transgene are GAC.

[0065] In some embodiments, at least 50% of the codons encoding glutamate residues in the transgene are GAG, optionally wherein about 57% of the codons encoding glutamate residues in the transgene are GAG.

[0066] In some embodiments, at least 50% of the codons encoding phenylalanine residues in the transgene are TTC, optionally wherein about 63% of the codons encoding phenylalanine residues in the transgene are TTC.

[0067] In some embodiments, at least 40% of the codons encoding glycine residues in the transgene are GGC, optionally wherein about 47% of the codons encoding glycine residues in the transgene are GGC, optionally wherein none of the codons encoding glycine residues are GGT.

[0068] In some embodiments, at least 50% of the codons encoding histidine residues in the transgene are CAC, optionally wherein about 62% of the codons encoding histidine residues in the transgene are CAC.

[0069] In some embodiments, at least 40% of the codons encoding isoleucine residues in the transgene are ATC, optionally wherein about 66% of the codons encoding isoleucine residues in the transgene are ATC, optionally wherein none of the codons encoding isoleucine residues are ATA.

[0070] In some embodiments, at least 40% of the codons encoding lysine residues in the transgene are AAG, optionally wherein about 55% of the codons encoding lysine residues in the transgene are AAG.

[0071] In some embodiments, at least 50% of the codons encoding leucine residues in the transgene are CTG, optionally wherein all of the codons encoding leucine residues in the transgene are CTG.

[0072] In some embodiments, at least 30% of the codons encoding asparagine residues in the transgene are AAC, optionally wherein about 54% of the codons encoding asparagine residues in the transgene are AAC.

[0073] In some embodiments, at least 45% of the codons encoding proline residues in the transgene are CCA, optionally wherein about 48% of the codons encoding proline residues in the transgene are CCA, optionally wherein none of the codons encoding proline residues in the transgene are CCG.

[0074] In some embodiments, at least 60% of the codons encoding glutamine residues in the transgene are CAG, optionally wherein all of the codons encoding glutamine residues in the transgene are CAG. In some embodiments, at least 50% of the codons encoding arginine residues in the transgene are CGG, CGA, or CGC, optionally wherein none of the codons encoding arginine residues in the transgene are CGT.

[0075] In some embodiments, at least 60% of the codons encoding serine residues in the transgene are AGT, AGC, or TCC and / or less than 20% of the codons encoding serine residues in the transgene are TCA, optionally wherein none of the codons encoding serine residues in the transgene are TCG.

[0076] In some embodiments, at least 20% of the codons encoding threonine residues in the transgene are ACC, optionally wherein about 40% of the codons encoding threonine residues in the transgene are ACC, optionally wherein none of the codons encoding threonine residues in the transgene are ACG.

[0077] In some embodiments, at least 60% of the codons encoding valine residues in the transgene are GTG or GTC, optionally wherein none of the codons encoding valine residues in the transgene are GT A or GTT.

[0078] In some embodiments, at least 30% of the codons encoding tyrosine residues in the transgene are TAC, optionally wherein about 50% of the codons encoding tyrosine residues in the transgene are TAC.

[0079] RPGR polypeptide

[0080] The human RPGR gene is located in chromosomal region Xp21.1 and spans 172 kb. There are multiple alternatively spliced transcripts, all of which encode an amino (N)-terminal RCCl-like (RCCL) domain. The RCCL domain is structurally similar to the RCC1 protein, a guanine nucleotide exchange factor for the small guanosine triphosphate-binding protein, Ran. The RPGR gene contains 19 exons (RPGRexl-19), encoding a predicted 90 kDa protein. Exons 2 to 11 encode the RCCL domain, whereas exons 12 to 19 encode a carboxyl (C)-terminal domain rich in acidic residues and ending in an isoprenylation anchorage signal. Mutations found in RPGRexl-19 account for 15% to 20% of XLRP patients, and subsequent studies revealed many more disease-causing mutations within one or more transcripts containing an alternatively spliced C-terminal exon called ORF 15 (RPGR ORF 15). A high frequency of microdeletions, frameshift, and premature stop mutations are found within the ORF 15. Without wishing to be bound by theory, it is believed that the use of full-length RPGR ORF 15 in a gene therapy vector improves the efficacy of said vector in

[0081] the treatment of XLRP.

[0082] In some embodiments, the transgene comprises RPGR ORF 15. In some embodiments, the transgene encodes an RPGR polypeptide of at least 550 amino acids, optionally the transgene encodes an RPGR polypeptide comprising 567 amino acids. In some embodiments, the transgene encodes an RPGR polypeptide of at least 1100 amino acids, optionally the transgene encodes an RPGR polypeptide comprising 1152 amino acids. In some embodiments, the transgene encodes the full-length human RPGR ORF 15 polypeptide, for example the RPGR ORF 15 polypeptide of SEQ ID NO: 11. By ‘full-length” it is meant that the transgene encodes all of the amino acids of the native RPGR ORF 15 polypeptide, i.e. the full-length RPGR ORF 15 polypeptide does not comprise any deletions or truncations and is not ‘abbreviated’, or the transgene encoding the full-length RPGR ORF 15 polypeptide does not comprise any deletions of codons coding for the RPGR ORF 15 polypeptide.

[0083] In some embodiments, the transgene comprises at least 3100, at least 3200, at least 3300, or at least 3400 nucleotides.

[0084] Codon optimisation and CpGs

[0085] In some embodiments, the transgene and / or the polynucleotide is codon-optimised.

[0086] Codon-optimisation can improve expression of the encoded product from the nucleotide sequence, for example an RPGR nucleotide sequence, in a particular cell type, tissue and / or in a particular organism. For example, if a nucleotide sequence is codon-optimised for expression in human cells, the nucleotide sequence may be modified to increase the number of codons that may be favoured (in the sense that such codons may correspond to tRNA species which are more abundant than other tRNA species specific for the same amino acid) in human cells. As a further example, if a nucleotide sequence is codon-optimised for expression in human cells, the nucleotide sequence may be modified to increase the number of codons that may be favoured in human cells. The skilled person would appreciate that codon-optimising a sequence may not entail changing every codon, not least because a "'favoured codon” may already be present at some positions.

[0087] In some embodiments, the transgene and / or the polynucleotide is codon optimised if the product of the RPGR transgene is expressed at a higher level in HEK293T cells compared to an equivalent polynucleotide comprising a corresponding RPGR transgene. For example, the transgene and / or the polynucleotide is codon optimised if the product of the RPGR transgene is expressed at a level that is a factor of at least 1.5, or a factor of at least 2 better in HEK293T cells compared to an equivalent polynucleotide comprising a corresponding RPGR transgene. An equivalent polynucleotide is identical to the test polynucleotide but is not codon optimised. A corresponding RPGR transgene is identical to the test RPGR transgene but comprises a wild-type RPGR sequence.

[0088] In some embodiments, a transgene of the invention may comprise more CpGs (i.e. CG dinucleotides) than a wild-type RPGR transgene. For example, the transgene may comprise more than 161 CpGs. In some embodiments, the transgene comprises about 167 CpGs. For example, there are 167 CG dinucleotides in SEQ ID NO: 2.

[0089] hGRKl promoter

[0090] A polynucleotide of the invention comprises an hGRKl promoter.

[0091] hGRKl promoters are derived from the promoter of the human Rhodopsin kinase gene. hGRKl promoters are described in Young et al., A short, highly active photoreceptor-specific enhancer / promoter region upstream of the human rhodopsin kinase gene, Sept 2003, Investigative Ophthalmology & Visual Science, 44:4076-4085 and Khani et al., AAV-mediate expression targeting of rod and cone photoreceptors with a human rhodopsin kinase promoter, Sept 2007, Investigative Ophthalmology & Visual Science, 48:3954-3961. For example, an hGRKl promoter may be the nucleotide sequence at positions -112 to +87 of the hGRKl gene, the nucleotide sequence at positions -112 to +180, or a nucleotide sequence derived from one of these sequences.

[0092] In some embodiments, the hGRKl promoter comprises a sequence that is between 200 and 300 nucleotides in length. In some embodiments, the hGRKl promoter comprises a sequence that is between 250 and 300 nucleotides in length. In some embodiments, the hGRKl is around 295 nucleotides in length. For the purposes of the present invention, the term "around' refers to within 1% of, i.e. a nucleotide sequence will be around 295 nucleotides in length if it is between 293 and 297 nucleotides in length. In some embodiments, the hGRKl promoter is at least 293 nucleotides in length. In some embodiments, the hGRKl promoter is between 293 and 297 nucleotides in length. In some embodiments, the hGRKl promoter is between 293 and 296 nucleotides in length. In some embodiments, the hGRKl promoter is 295 nucleotides in length.

[0093] In some embodiments, the hGRKl promoter comprises a sequence that is identical to SEQ ID NO: 10, but for:

[0094] (a) a T to G substitution at position 199;

[0095] (b) a C to A substitution at position 245; and / or

[0096] (c) a GGC insertion between positions 246 and 247.

[0097] In some embodiments, the hGRKl promoter comprises a sequence that is identical to SEQ ID NO: 10, but for 2 of:

[0098] (a) a T to G substitution at position 199;

[0099] (b) a C to A substitution at position 245; and / or

[0100] (c) a GGC insertion between positions 246 and 247.

[0101] In some embodiments, the hGRKl promoter comprises a sequence that is identical to SEQ ID NO: 10, but for a T to G substitution at position 199, and a C to A substitution at position 245. In some embodiments, the hGRKl promoter comprises a sequence that is identical to SEQ ID NO: 10, but for a T to G substitution at position 199 and a GGC insertion between positions 246 and 247. In some embodiments, the hGRKl promoter comprises a sequence that is identical to SEQ ID NO: 10, but for a C to A substitution at position 245 and a GGC insertion between positions 246 and 247. In some embodiments, the hGRKl promoter comprises a sequence that is identical to SEQ ID NO: 10, but for a T to G substitution at position 199, a C to A substitution at position 245 and a GGC insertion between positions 246 and 247.

[0102] In some embodiments, the hGRKl promoter comprises a sequence at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 1. In some embodiments, the hGKRl promoter comprises a sequence at least 99.5% identical to SEQ ID NO: 1. In some embodiments, the hGRKl promoter comprises a sequence that is identical to SEQ ID NO: 1.

[0103] In some embodiments, the hGRKl promoter comprises a sequence that is identical to SEQ ID NO: 1, except that the nucleotide corresponding to position 288 or 289 is mutated and / or a nucleotide is inserted after the nucleotide corresponding to position 227, 228 or 229.

[0104] Optionally, the nucleotide corresponding to position 288 or 289 is deleted. Optionally, a G nucleotide is inserted after the nucleotide corresponding to position 227, 228 or 229.

[0105] In some embodiments, the hGRKl promoter comprises a sequence at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 17. In some embodiments, the hGKRl promoter comprises a sequence at least 99.5% identical to SEQ ID NO: 17. In some embodiments, the hGRKl promoter comprises a sequence that is identical to SEQ ID NO: 17.

[0106] The hGRKl promoter may be able to promote gene expression at a level at least 90% of the level promoted by a promoter of SEQ ID NO: 1. Optionally, whether or not an hGRKl promoter is able to promote gene expression at a level at least 90% of the level promoted by a promoter of SEQ ID NO: 1 is measured by comparing expression of the hGRKl promoter to the promoter of SEQ ID NO: 1 in an expression assay comprising:

[0107] (i) transfecting HEK293T cells with a plasmid comprising an expression cassette comprising a transgene operably linked to the hGRKl promoter or promoter of SEQ ID NO:1;

[0108] (ii) incubating the transfected cells under conditions suitable for transgene expression to occur; and (iii) measuring the level of the transgene by ELISA using an antibody specific for a protein encoded by the transgene.

[0109] A transgene is “operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or transcription regulatory sequence is operably linked to a transgene if it affects the transcription of the transgene. Optionally, the promoter or transcription regulatory sequence is 5’ of the transgene. Optionally, the promoter is immediately 5’ of the transgene, or it is separated from the transgene by another sequence such as an intron. In some embodiments, the hGRKl promoter is operably linked to the transgene. Optionally, the hGRKl promoter is 5’ of an SV40 intron which is 5’ of the transgene. Optionally, the hGRKl promoter is immediately 5’ of an SV40 intron which is immediately 5’ of the transgene.

[0110] Optionally, the transgene is operably linked to a promoter if the promoter promotes expression at least 90% of the level of expression of the same promoter immediately upstream of the same transgene. Optionally, expression is measured by an expression assay comprising:

[0111] (i) transfecting HEK293T cells with a plasmid comprising an expression cassette comprising the transgene operably linked to the promoter or an equivalent expression cassette in which the promoter is immediately upstream of the transgene;

[0112] (ii) incubating the transfected cells under conditions suitable for transgene expression to occur; and

[0113] (iii) measuring the level of the transgene by ELISA using an antibody specific for a protein encoded by the transgene.

[0114] Inverted terminal repeats (ITRs)

[0115] The term “inverted terminal repeat” (ITR) refers to a nucleotide sequence located at the 5’ end (5TTR) and a nucleotide sequence located at the 3’end (3TTR) of a viral genome. An ITR comprises palindromic sequences and can fold over to form T-shaped hairpin structures that function as primers during initiation of DNA replication, in addition to performing other functions. The ITRs are required in cis for the vector genome replication and its packaging into the viral particles.

[0116] The polynucleotide of the invention comprises two ITRs. In some embodiments, the ITRs comprise a 5’ ITR and a 3’ ITR.

[0117] In some embodiments, the ITRs are from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV 8, AAV9, or AAV10. In some embodiments of the invention, the ITRs are from AAV2 or AAV5. In some embodiments of the invention, the ITRs are from AAV2.

[0118] In some embodiments, the 5’ ITR comprises a sequence:

[0119] (a) at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 3; or

[0120] (b) that is identical to SEQ ID NO: 3.

[0121] In some embodiments, the 5’ UTR comprises a sequence at least 98% identical to SEQ ID NO: 3. In some embodiments, the 5’ UTR comprises a sequence that is identical to SEQ ID NO: 3.

[0122] In some embodiments, the 3’ ITR comprises a sequence:

[0123] (a) at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 4; or

[0124] (b) that is identical to SEQ ID NO: 4.

[0125] In some embodiments, the 3’ UTR comprises a sequence at least 98% identical to SEQ ID NO: 4. In some embodiments, the 3’ ITR comprises a sequence that is identical to SEQ ID NO: 4.

[0126] Introns

[0127] In the context of a viral vector, an intron is a non-coding section of DNA which may direct or influence transcription of a transgene in which it is present. In some cases, an intron is removed from an mRNA molecule through a splicing event after transcription. In some cases, an intron is located between a transcription regulatory element and the coding nucleotide sequence of a transgene.

[0128] The polynucleotide of the invention comprises an SV40 late intron. In some embodiments, the SV40 late intron comprises a sequence at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 5. In some embodiments, the SV40 late intron comprises a sequence at least 98% identical to SEQ ID NO: 5. In some embodiments, the SV40 late intron comprises a sequence identical to SEQ ID NO: 5.

[0129] In some embodiments, the SV40 late intron comprises a sequence at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 12. In some embodiments, the SV40 late intron comprises a sequence at least 98% identical to SEQ ID NO: 12. In some embodiments, the SV40 late intron comprises a sequence identical to SEQ ID NO: 12.

[0130] In some embodiments, the SV40 late intron is operably linked to the transgene. Optionally, the SV40 late intron is 5’ of the transgene. Optionally, the SV40 late intron is immediately 5’ of the transgene.

[0131] PolyA

[0132] As used herein, the term “ polyadenylation tail” (or polyA) refers to a specific recognition sequence within the 3’ untranslated region (3’ UTR) of the gene, which is transcribed into a precursor mRNA molecule and guides the termination of gene transcription. The polyA sequence acts as a signal for the endonucleolytic cleavage of the newly formed precursor mRNA at its 3 ’-end, and for the addition of an RNA stretch consisting only of adenine bases to the 3’ -end. This is known as polyadenylation. The polyA tail is important for nuclear export, translation, and stability of mRNA.

[0133] The polynucleotide of the invention comprises an SV40 late polyA. In some embodiments, the SV40 late polyA comprises a sequence at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 6. In some embodiments, the SV40 late polyA comprises a sequence at least 98% identical to SEQ ID NO: 6. In some embodiments, the SV40 late polyA comprises a sequence identical to SEQ ID NO: 6.

[0134] In some embodiments, the SV40 late polyA comprises a sequence at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 13. In some embodiments, the SV40 late polyA comprises a sequence at least 98% identical to SEQ ID NO: 13. In some embodiments, the SV40 late polyA comprises a sequence identical to SEQ ID NO: 13.

[0135] Polynucleotide

[0136] In some embodiments, the polynucleotide of the invention comprises the following elements in 5’ to 3’ order: the 5’ ITR, the hGRKl promoter, the SV40 late intron, the transgene encoding the RPGR polypeptide, the SV40 late polyA, and the 3’ ITR. In some embodiments, the 5’UTR is immediately 5’ of the hGRKl promoter. In some embodiments, the hGRKl promoter is immediately 5’ of the SV40 late intron. In some embodiments, the SV40 late intron is immediately 5’ of the transgene encoding the RPGR polypeptide. In some embodiments, the transgene encoding the RPGR polypeptide is immediately 5’ of the SV40 late polyA. In some embodiments, the SV40 late polyA is immediately 5’ of the 3’ UTR. In some embodiments, there are no intervening nucleotide sequences, i.e. the 5’ ITR is immediately 5’ of the hGRKl promoter, the hGRKl promoter is immediately 5’ of the SV40 late intron, the SV40 late intron is immediately 5’ of the transgene encoding the RPGR polypeptide, the transgene is immediately 5’ of the SV40 late polyA, and the SV40 late polyA is immediately 5’ of the 3’ ITR.

[0137] In some embodiments, the polynucleotide is:

[0138] (a) between 4000 and 5000 nucleotides in length;

[0139] (b) between 4000 and 4500 nucleotides in length;

[0140] (c) between 4250 and 4500 nucleotides in length;

[0141] (d) less than 4500 nucleotides in length; or

[0142] (e) around 4500 nucleotides in length. In some embodiments, the polynucleotide is between 4250 and 4500 nucleotides in length. In some embodiments, the polynucleotide is around 4500 nucleotides in length.

[0143] In some embodiments, the polynucleotide comprises a nucleotide sequence:

[0144] (a) at least 90%, at least 95%, or at least 98% identical to a fragment of at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, or at least 4250 nucleotides of SEQ ID NO: 8;

[0145] (b) at least 95% identical to a fragment of at least 4250 nucleotides of SEQ ID NO: 8; (c) at least 98% identical to a fragment of at least 4250 nucleotides of SEQ ID NO: 8; (d) at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 8;

[0146] (e) at least 98% identical to SEQ ID NO: 8; and / or

[0147] (f) identical to SEQ ID NO: 8.

[0148] In some embodiments, the polynucleotide comprises a sequence at least 98% identical to a fragment of at least 4250 nucleotides of SEQ ID NO: 8. In some embodiments, the polynucleotide comprises a sequence at least 98% identical to SEQ ID NO: 8. In some embodiments, the polynucleotide comprises a sequence identical to SEQ ID NO: 8.

[0149] Viral particle

[0150] The term “viral particle" relates to a typically replication-defective virus particle comprising (i) preferably at least a portion of a viral genome (ii) a capsid and optionally (but not in the case of AAV), (iii) a lipidic envelope surrounding the capsid. The term “viral particle" includes recombinant adeno-associated viral (AAV) particles, comprising an AAV capsid and a genome comprising sequences designed to function in an AAV (like ITR sequences).

[0151] The term “viral genome'' refers to the nucleic acid part of the viral particle disclosed herein, which may be packaged in a capsid. In the present invention, the viral particle may be a recombinant adeno-associated viral particle (rAAV). The terms rAAV and AAV are used interchangeably herein, unless the context indicates otherwise.

[0152] The genomic organization of all known AAV serotypes is very similar. The genome of native (non-recombinant) AAV is a linear, single-stranded DNA molecule that is less than about 5,000 nucleotides in length. Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences for the non- structural replication (Rep) proteins and the structural (VP) proteins. The VP proteins (VP1, -2 and -3) form the capsid. The terminal 145 nt are self-complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These hairpin structures function as an origin for viral DNA replication, serving as primers for the cellular DNA polymerase complex.

[0153] Following wild type (wt) AAV infection in mammalian cells the Rep genes (i.e. encoding Rep78 and Rep52 proteins) are expressed from the P5 promoter and the P19 promoter, respectively, and both Rep proteins have a function in the replication of the vector genome. A splicing event in the Rep ORF results in the expression of four Rep proteins (i.e. Rep78, Rep68, Rep52 and Rep40). However, it has been shown that the unspliced mRNA, encoding Rep78 and Rep52 proteins, in mammalian cells are sufficient for AAV vector production. Also in insect cells the Rep78 and Rep52 proteins suffice for AAV vector production.

[0154] An AAV particle of the present invention may lack genes encoding the VP proteins and may lack genes encoding the Rep proteins. Indeed, AAV particles of the present invention may comprise a recombinant genome lacking all AAV genes, except (for example) they may comprise nucleic acid material comprising a 5’ ITR and a 3’ ITR.

[0155] In some embodiments, the present invention relates to a viral particle comprising a recombinant genome (i.e. the viral genome) comprising the polynucleotide of the invention. In some embodiments, the viral particle is an AAV, adenoviral, or lentiviral particle. In a preferred embodiment, the viral particle is an AAV particle.

[0156] In some embodiments, the viral particle is capable of delivering the transgene to ocular cells. In some embodiments the ocular cells are retinal and / or para-retinal cells, optionally including one or more cell types selected from retinal ganglion cells, amacrine cells, horizontal cells, bipolar cells, photoreceptor cells, cone cells, rod cells, Muller glial cells, retinal pigmented epithelium (RPE) cells and / or choroid cells. In some embodiments, the ocular cells are cells in the retinal pigment epithelial layer or cells in the choroidal capillary layer. In some embodiments the ocular cells are anterior ocular cell types such as cells of the ciliary body and / or iris, optionally in addition to the aforementioned retinal and para-retinal cells.

[0157] Optionally, the viral particle was produced in or is producible in mammalian cells. Optionally, the viral particle was not produced in and / or is not producible in insect cells. Optionally, the viral particle has features characteristic of viral particles produced in mammalian cells.

[0158] Optionally, the viral particle comprises proteins that have a pattern of post translational modifications characteristic of a viral particle that was produced in a mammalian cell.

[0159] AA V capsid

[0160] The viral particle of the invention comprises a capsid.

[0161] AAV capsids are generally formed from three proteins, VP1, VP2 and VP3. The amino acid sequence of VP1 comprises the sequence of VP2. The amino acid sequence of VP2 comprises the sequence of VP3. Differences among the capsid protein sequences of the various native and engineered AAV serotypes result in the use of different cell surface receptors for cell entry. In combination with alternative intracellular processing pathways, this gives rise to variation in tissue tropisms between the AAV serotypes.

[0162] In some embodiments, the capsid is an AAV5 capsid. In some embodiments, the capsid is not an AAV5 capsid. Optionally, the AAV capsid comprises an amino acid sequence:

[0163] (a) at least 90%, at least 95%, or at least 98% identical to a fragment of at least 600, at least 650, at least 675, or at least 700 amino acids of SEQ ID NO: 14;

[0164] (b) at least 95% identical to a fragment of at least 650 amino acids of SEQ ID NO: 14; (c) at least 98% identical to a fragment of at least 700 amino acids of SEQ ID NO: 14; or (d) at least 90%, at least 95%, at least 98%, at least 99% or at least 99.5% identical to SEQ ID NO: 14.

[0165] In some embodiments, the capsid is an AAV2 capsid, or an AAV2-derived capsid. In some embodiments, the capsid is an AAV2 capsid comprising a substitution of at least one Y residue with an F residue, optionally wherein the AAV2 capsid comprises a Y444F, Y500F, and / or Y730F substitution.

[0166] In some embodiments, the capsid comprises an amino acid sequence:

[0167] (a) at least 90%, at least 95%, or at least 98% identical to a fragment of at least 600, at least 650, at least 675, or at least 700 amino acids of SEQ ID NO: 9;

[0168] (b) at least 95% identical to a fragment of at least 650 amino acids of SEQ ID NO: 9; (c) at least 98% identical to a fragment of at least 700 amino acids of SEQ ID NO: 9; or

[0169] (d) at least 90%, at least 95%, at least 98%, at least 99% or at least 99.5% identical to SEQ ID NO: 9.

[0170] In some embodiments, the capsid comprises an amino acid sequence at least 99.5% identical to SEQ ID NO: 9. In some embodiments, the capsid comprises an amino acid sequence identical to SEQ ID NO: 9.

[0171] Composition

[0172] The invention provides a composition comprising a multiplicity of the viral particles of the invention, e.g. the composition may comprise 7.5 x 1010, 3.7 x 1011or 6.8 x 1011viral particles.

[0173] It is an inherent characteristic of the AAV manufacturing process that a portion of the viral particles produced will comprise capsids that are not packaged with the therapeutic transgene and are therefore referred to as “empty” capsids or particles, as opposed to so-called “full” capsids or particles which do contain the transgene. Viral particles may therefore be full or empty, or otherwise fall within a heterogeneous population of partially-filled f partial") capsids containing packaged process-related impurities or truncated genomes. A full viral particle contains within its capsid a recombinant genome comprising the therapeutic transgene, while an empty viral particle either does not contain a recombinant genome within its capsid or does not contain a transgene-carrying recombinant genome within its capsid. A viral particle of the invention is a full viral particle. However, it will be understood by the skilled person that when producing a composition comprising viral particles, not all of the particles will be full viral particles (have “full” capsids), i.e. the composition will comprise full and empty viral particles (capsids), as well as partials. For example, capsids may be assembled without a recombinant genome being packaged inside.

[0174] Full, empty and partial viral particles or capsids can be distinguished by their molecular weights, due to the presence or absence of the recombinant genome. As is known in the art, the skilled person can measure the proportion of full viral particles within a composition using various techniques, including sedimentation velocity analytical ultracentrifugation (SV-AUC) (see e.g. Werle AK, Powers TW, Zobel JF, Wappelhorst CN, Jarrold MF, Lyktey NA, Sloan CDK, Wolf AJ, Adams-Hall S, Baldus P, Runnels HA. Comparison of analytical techniques to quantitate the capsid content of adeno-associated viral vectors. Mol Ther Methods Clin Dev. 2021 Sep l;23:254-262). The proportion of full viral particles can be expressed either as a percentage of full viral particles (or capsids) or a so called full:empty capsid ratio, which is the proportion of the total viral particles which are full. The proportion of empty and partial viral particles or capsids can also be expressed either as a percentage of empty and partial viral particles, i.e. a percentage of viral particles containing within their respective capsids the recombinant genome comprising the transgene, or a so called empty:full capsid ratio, which is the proportion of the total viral particles which are empty or partial.

[0175] Without wishing to be bound by theory, it is believed that compositions with a higher percentage of full viral particles or capsids are safer and / or more effective for use in methods of treatment by gene therapy. For example, empty and partial viral particles may contribute to an immune response (by increasing the total titre of viral particles dosed), without providing therapeutic benefit due to the absence of the therapeutic transgene. A higher full:empty capsid ratio may result in greater expression of the transgene for a given dose (as measured by the particle / capsid titre), leading to an increased therapeutic effect. It is therefore desirable to provide compositions with a higher full:empty capsid ratio.

[0176] In some embodiments, the composition has:

[0177] (a) a percentage of viral particles containing within their respective capsids the recombinant genome comprising the transgene that is greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90%;

[0178] (b) a percentage of viral particles comprising full capsids that is greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90%; and / or

[0179] (c) a full: empty capsid ratio that is greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90%.

[0180] In some embodiments, greater than 70% of the viral particles comprise a recombinant genome.

[0181] In some embodiments, the composition has:

[0182] (a) a percentage of viral particles not containing within their respective capsids the recombinant genome comprising the transgene that is less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10%;

[0183] (b) a percentage of viral particles not comprising full capsids that is less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10%; and / or

[0184] (c) an empty:full capsid ratio that is less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10%.

[0185] In some embodiments, less than 30% of the viral particles lack a recombinant genome.

[0186] In some embodiments, the percentage and / or full: empty capsid ratio is measured by SV-AUC. In this context, SV-AUC measures the difference in buoyant density between empty and full capsids. Full capsids have a greater buoyant density than empty capsids and, therefore, sediment more quickly through solution due to differential sedimentation coefficients (c(s)). An AUC c(s) distribution plot will show distinct peaks corresponding to empty and full capsids, as well as an additional intervening peak corresponding to partials. Because the nucleic acid content of the capsid influences the absorbance at A260 and A280, the resulting A260 / A280 ratio for each species in the c(s) plot of the absorbance data can be used to support the identification of peaks in the c(s) plot. The A260 / A280 ratio is obtained by integrating the peak area of individual species in the c(s) plot at both wavelengths and dividing the A260 measurement by the A280 measurement. In this way, as evident in the art (from e.g. Werle AK, Powers TW, Zobel JF, Wappelhorst CN, Jarrold MF, Lyktey NA, Sloan CDK, Wolf AJ, Adams-Hall S, Baldus P, Runnels HA. Comparison of analytical techniques to quantitate the capsid content of adeno-associated viral vectors. Mol Ther Methods Clin Dev. 2021 Sep l;23:254-262) the skilled person is readily able to calculate the relative proportions of full, empty and partial capsids.

[0187] In some embodiments, there is provided a composition comprising the polynucleotide or viral particle of the invention and a pharmaceutically acceptable excipient. The term "pharmaceutically acceptable" means approved by a regulatory agency or recognized pharmacopeia such as European Pharmacopeia, for use in animals and / or humans. The term "excipient" refers to a diluent, adjuvant, carrier, or vehicle with which the therapeutic agent is administered.

[0188] Any suitable pharmaceutically acceptable carrier, diluent or excipient can be used in the preparation of a pharmaceutical composition (See e.g., Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro (Editor) Mack Publishing Company, April 1997).

[0189] Pharmaceutical compositions are typically sterile and stable under the conditions of manufacture and storage. Pharmaceutical compositions may be formulated as solutions (e.g. saline, dextrose solution, or buffered solution, or other pharmaceutically acceptable sterile fluids), microemulsions, liposomes, or other ordered structure suitable to accommodate a high product concentration (e.g. microparticles or nanoparticles). The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, or salts such as sodium chloride in the composition.

[0190] In some embodiments, the composition further comprises:

[0191] (a) sodium chloride;

[0192] (b) potassium chloride;

[0193] (c) calcium chloride dihydrate;

[0194] (d) magnesium chloride hexahydrate;

[0195] (e) sodium acetate trihydrate;

[0196] (f) sodium citrate dihydrate;

[0197] (g) sodium hydroxide;

[0198] (h) hydrochloric acid; and / or

[0199] (i) polysorbate 20 (v / v).

[0200] In some embodiments, the composition further comprises at least 2 of (a) to (i) in the preceding paragraph. In some embodiments, the composition further comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or 9 of excipients (a) to (i) in the preceding paragraph. In some embodiments, the composition further comprises all of excipients (a) to (h) in the preceding paragraph. In some embodiments, the composition comprises excipients (a), (b), (c), (d), (e), (f), and (i) in the preceding paragraph.

[0201] In some embodiments, the composition comprises:

[0202] (a) equal to or greater than 2.5 x 1011vg / ml of the viral particles;

[0203] (b) between 6 and 7 mg / ml sodium chloride;

[0204] (c) between 0.5 and 1 mg / ml potassium chloride;

[0205] (d) between 0.4 and 0.6 mg / ml calcium chloride dihydrate;

[0206] (e) between 0.2 and 0.4 mg / ml magnesium chloride hexahydrate;

[0207] (f) between 3.5 and 4.5 mg / ml sodium acetate trihydrate;

[0208] (g) between 1.5 and 2 mg / ml sodium citrate dihydrate; and / or

[0209] (h) between 0.01 and 0.02% polysorbate 20 (v / v). In some embodiments, the composition further comprises at least 2 of (a) to (i) in the preceding paragraph. In some embodiments, the composition further comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8 of (a) to (i) in the preceding paragraph. In some embodiments, the composition further comprises all of (a) to (h) in the preceding paragraph.

[0210] Methods of medical treatment / medical uses

[0211] The terms “treatment”, “treating” and the like refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptoms thereof from appearing or worsening and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease.

[0212] In some embodiments of the invention, treatment of an ocular disease, such as X-linked retinitis pigmentosa (XLRP), may comprise an improvement in the quality of vision in a subject, or a slowing or arresting of progression of deterioration of vision. For example, treatment of an ocular disease may comprise a reduction in blurry or fuzzy vision (an improvement in visual acuity), reduction or elimination of a blind spot in the centre of vision or the progression of such a blind spot, and / or reduction in the extent or pace of loss of peripheral vision and / or night vision or an improvement in these characteristics. Treatment of an ocular disease, such as XLRP, may comprise an increase in fine detail of vision, an improvement in the ability to perceive faces, or an improvement in the ability to read.

[0213] Treatment of an ocular disorder may be measured with a visual acuity test, such as a best corrected visual acuity (BCVA) or a low luminance visual acuity (LLVA) test (for LLVA, see Examples and Wood LJ, Jolly JK, Buckley TMW, Josan AS, & MacLaren RE. Low luminance visual acuity as a clinical measure and clinical trial outcome measure: a scoping review. Ophthalmic Physiol Opt. 2021; 41: 213-223). A visual acuity test is a functional test that measures the ability of an eye of a subject, e.g. a treated eye, to discern letters or symbols of various sizes from a distance. BCVA and LLVA assessments typically use the standard Early Treatment Diabetic Retinopathy Study (ETDRS) chart, with BCVA performed in photopic (bright-light), and LLVA in mesopic (low-light), conditions. It is within the capabilities of the skilled person to perform a visual acuity test. In some embodiments, treatment of an ocular disorder comprises improving the performance of a treated eye of a subject in a visual acuity test such as LLVA.

[0214] In some embodiments, the treatment improves the performance of the treated eye of the subject in LLVA by at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 15, at least 16, at least 18, at least 20, at least 22, or at least 24 letters. In a preferred embodiment, the treatment improves the performance of the treated eye of the subject in LLVA by between 10 and 14 letters, or by at least 15 letters. In some embodiments, the improvement is seen at least 3, at least 6, at least 9, at least 12, at least 18, or at least 24 months after treatment. In some embodiments, the improvement is seen after 6 or 12 months. In some embodiments, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of treated eyes respond to the treatment by at least 3, at least 6, at least 9, at least 12, at least 18, or at least 24 months after treatment, wherein a treated eye responds to the treatment if the treatment improves the performance of the treated eye in LLVA by at least 10-14 or at least 15 letters. In some embodiments, at least 60% of the treated eyes respond to the treatment at least 12 months after treatment, wherein a treated eye responds to the treatment if the treatment improves the performance of the treated eye in LLVA by at least 10-14 or at least 15 letters.

[0215] In some embodiments, a greater improvement in the performance of a treated eye of a subject in LLVA may be seen in eyes with a higher low luminance deficit (LLD) at baseline (i.e. before treatment). LLD is calculated by taking the LLVA score of an eye away from the corresponding BCVA of the same eye. In some embodiments, the subject eye to be treated (i.e. the treated eye of the subject prior to treatment) according to the present invention has a baseline LLD of at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, or at least 24 letters. In some embodiments, the subject eye to be treated has a baseline LLD of at least 20 letters. In some embodiments, at least 80% of subject eyes to be treated with a baseline LLD of at least 20 letters respond to the treatment at least 12 months after treatment, wherein a treated eye of a subject responds to the treatment if the treatment improves the performance of the eye in LLVA by at least 10-14 letters or at least 15 letters. Treatment of an ocular disorder may be measured by a test of retinal sensitivity, optionally a microperimetry test which quantifies retinal sensitivity through the ability of an eye of a subject to detect light stimuli occurring across a grid of loci (see Examples and Yang Y, Dunbar H. Clinical Perspectives and Trends: Microperimetry as a Trial Endpoint in Retinal Disease. Ophthalmologica. 2021;244(5):418-450. doi: 10.1159 / 000515148. Epub 2021 Feb 10. PMID: 33567434; PMCID: PMC8686703). In some embodiments, treatment of an ocular disorder comprises improving the performance of a treated eye of a subject in a retinal sensitivity test such as microperimetry. In some embodiments, macular integrity assessment (MAIA) microperimetry is used to assess improvement of retinal sensitivity in a treated eye of a subject.

[0216] In some embodiments, the treatment increases the mean retinal sensitivity of a treated eye of a subject, as measured by microperimetry. In some embodiments, the treatment increases the mean retinal sensitivity of the treated eye by at least 0.5 dB, at least 1.0 dB, at least 1.5 dB, at least 2.0 dB, or by at least 2.5 dB. In some embodiments, the treatment increases the mean retinal sensitivity of the treated eye by at least 2.0 dB. In some embodiments, the treatment increases the retinal sensitivity of the treated eye by at least 7 dB at at least 5 loci. In some embodiments, the increase is seen at least 3, at least 6, at least 9, at least 12, at least 18, or at least 24 months after treatment. In some embodiments, the increase is seen 3 months after treatment. In some embodiments, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of treated eyes respond to the treatment by at least 3, at least 6, at least 9, at least 12, at least 18, or at least 24 months after treatment, wherein a treated eye of a subject responds to the treatment if the treatment increases the retinal sensitivity of the eye by at least 7 dB at at least 5 loci or increases the mean retinal sensitivity of the treated eye by at least 0.5 dB, at least 1.0 dB, at least 1.5 dB, at least 2.0 dB, or by at least 2.5 dB. In some embodiments, at least 60% of treated eyes respond to the treatment at least 12 months after treatment, wherein a treated eye responds to the treatment if the treatment increases the retinal sensitivity of the treated eye by at least 7 dB at at least 5 loci or increases the mean retinal sensitivity of the treated eye by at least 0.5 dB, at least 1.0 dB, at least 1.5 dB, at least 2.0 dB, or by at least 2.5 dB. Treatment of an ocular disorder may be measured by a mobility course (sometimes referred to as a ‘maze’), such as the Ora- Visual Navigation Challenge (VNC™), which functionally assesses a subject’s ability and speed at navigating a route whilst avoiding obstacles under defined conditions such as luminance level. In some embodiments, treatment of an ocular disorder comprises improving the performance of a subject in a mobility course such as the Ora- VNC™.

[0217] In some embodiments, the present invention relates to a method of treating an ocular disorder in a subject. In some embodiments, the method of treating an ocular disorder comprises a step of administration of a polynucleotide, viral particle, or composition of the invention. In some embodiments, the ocular disorder is X-linked retinitis pigmentosa (XLRP).

[0218] Dosage

[0219] In some embodiments, a single dose of the polynucleotide, viral particle, composition or pharmaceutical composition is administered to the eye of the subject. The dose is measured using vector genome (vg) numbers. In some embodiments, the polynucleotide, viral particle, composition or pharmaceutical composition is administered at a dose of around 7.5 x 1010, 3.7 x 1011or around 6.8 x 1011vg / eye. In some embodiments, the polynucleotide, viral particle, composition or pharmaceutical composition is administered at a dose of around 1 x 1011, 2 x 1011or 5 x 1010vg / eye.

[0220] Combination treatment with steroids

[0221] In some embodiments, the viral particle of the invention is administered in combination with a steroid. In some embodiments, the steroid is a corticosteroid. In some embodiments, the steroid comprises a steroid selected from the group consisting of dexamethasone, prednisolone, methylprednisolone and hydrocortisone. In some embodiments, the steroid comprises prednisolone. In some embodiments, the steroid is prednisone. In some embodiments, the steroid is administered systemically such as orally. In some embodiments, the steroid is administered topically, for example the steroid may be administered to the eye. In some embodiments, the steroid is administered via periocular administration, intravitreal administration, subtenons administration, subconjunctival administration, or superchoroidal administration. In some embodiments, the steroid is administered via a drop. In some embodiments, the steroid is a glucocorticoid, for example an anti-inflammatory glucocorticoid. In some embodiments, the steroid is difluprednate, medrysone, loteprednol, prednisolone, fluocinolone, triamcinolone, rimexelone, dexamethasone, fluoromethoIone, or prednisone.

[0222] In some embodiments, the steroid is administered before, during, and / or after administration of the viral particle. In some embodiments, the steroid is administered before administration of the viral particle. In some embodiments, the steroid is administered during (i.e. at the same time as) administration of the viral particle. In some embodiments, the steroid is administered after the viral particle.

[0223] Improvements

[0224] In some embodiments, the treatment improves visual function of the treated eye of the subject by more than treatment with a corresponding composition having a lower percentage of full capsids and / or fulkempty ratio. Optionally, visual function is assessed by measuring low luminance visual acuity (LLVA). Optionally, the treatment improves the performance of the treated eye of the subject in LLVA by at least 2, at least 4, at least 6, at least 8, at least 10 letters more than treatment with a corresponding composition having a lower percentage of viral particles comprising full capsids and / or a lower full:empty capsid ratio. In some embodiments, at least 20% more of the treated eyes respond to the treatment at least 12 months after treatment compared with eyes treated with a corresponding composition having a lower percentage of viral particles comprising full capsids and / or a lower full:empty capsid ratio, wherein a treated eye responds to the treatment if the treatment improves the performance of the eye in LLVA by at least 10-14 letter or at least 15 letters.

[0225] In some embodiments, the treatment improves retinal sensitivity of the treated eye of the subject by more than treatment with a corresponding composition having a lower percentage of viral particles comprising full capsids and / or a lower fulkempty ratio. Optionally, the retinal sensitivity is measured by microperimetry such as MAIA microperimetry. Optionally, the treatment improves retinal sensitivity of the treated eye of the subject by at least 1 dB, at least 2 dB, at least 3 dB, at least 4 dB, at least 5 dB, at least 6 dB, at least 7 dB, at least 8 dB, at least 9 dB, or at least 10 dB more than treatment with a corresponding composition having a lower percentage of viral particles comprising full capsids and / or a lower full: empty ratio. In some embodiments, at least 20% more treated eyes respond to the treatment at least 12 months after treatment compared with eyes treated with a corresponding composition having a lower percentage of viral particles comprising full capsids and / or full: empty ratio, wherein a treated eye responds to the treatment if the treatment increases the retinal sensitivity of the treated eye by at least 7 dB at at least 5 loci or increases the mean retinal sensitivity of the treated eye by at least 0.5 dB, at least 1.0 dB, at least 1.5 dB, at least 2.0 dB, or by at least 2.5 dB.

[0226] In some embodiments, the treatment shows the same or greater improvement in visual function than treatment with a composition comprising viral particles comprising the vector genome of SEQ ID NO: 8 and the capsid of SEQ ID NO: 9, wherein the composition: (a) was produced by a downstream manufacturing process comprising an anion exchange (AEX) chromatography step or (b) has a full: empty capsid ratio of at least 70%.

[0227] In some embodiments, the treatment has an improved safety profile, such as fewer adverse events or serious adverse events, than treatment with a corresponding composition having a lower percentage of viral particles comprising full capsids and / or full: empty capsid ratio. In some embodiments, the treatment shows the same or fewer adverse effects and / or serious adverse effects than treatment with a composition comprising viral particles comprising the vector genome of SEQ ID NO: 8 and the capsid of SEQ ID NO: 9, wherein the composition: (a) was produced by a downstream manufacturing process comprising an anion exchange (AEX) chromatography step or (b) has a full: empty capsid ratio of at least 70%.

[0228] In some embodiments, the improvements in visual function or safety profile may be more pronounced when a lower dose of AAV particle is used. For example, where the dose is less than 5 x 1011vg (e.g. 7.5 x 1010or 3.7 x 1011), treatment using a composition having a full:empty capsid ratio of at least 70% may improve retinal sensitivity by at least 3 dB more than treatment with a corresponding composition having a lower percentage of viral particles comprising full capsids and / or a lower full: empty ratio (e.g. a full: empty ratio of less than 70%, such as 50%).

[0229] AA V production

[0230] The AAV particles according to the present invention may be produced by means of conventional methods and protocols. For example, the AAV particles may be produced using a recombinant herpes simplex virus (rHSV) complementation system. The system employs two rHSV helper viruses that are replication-incompetent due to a deletion in the UL54 (ICP27) gene. These ICP27 mutant viruses are replication-incompetent in normal cells but can be propagated in V27 cells (Vero cells stably transformed with the UL54 gene). They are doubly replication-incompetent in vivo, because of a deletion in the UL54 gene and also because of an insertion in the thymidine kinase gene (tk gene).

[0231] Two rHSV viruses are necessary for rAAV DNA replication and packing. One rHSV helper virus contains the AAV rep and cap genes and the second contains a cDNA expression cassette for a therapeutic gene of interest flanked by the AAV inverted terminal repeats. When used to infect serum-free suspension baby hamster kidney (SF-sBHK) cells, these two rHSV helper viruses together provide all cis- and trans-acting AAV components, as well as the requisite helper functions necessary for efficient rAAV production.

[0232] rHSV complementation systems are described in Clement et al., Manufacturing of recombinant adeno-associated viral vectors for clinical trials, Molecular Therapy — Methods & Clinical Development (2016) 3, 16002; Thomas et al., Scalable recombinant adeno-associated virus production using recombinant herpes simplex virus type 1 coinfection of suspension-adapted mammalian cells, Human Gene Therapy (2009) 20:861-870; Clement et al., Large-scale adeno-associated viral vector production using a herpesvirus-based system enables manufacturing for clinical studies, Human Gene Therapy (2009) 20:796-806. The former article (Clement, 2016) also discusses other well-known means of producing rAAV, including by plasmid transfection in mammalian cell lines, baculovirus-mediated production in insect cells, and mammalian producer cells lines.

[0233] 37 Purification methods

[0234] As set out above, it is advantageous to provide compositions comprising viral particles having a high full: empty capsid ratio. The present inventors have developed a purification protocol comprising a step of anion exchange (AEX) chromatography that leads to an increase in the full:empty capsid ratio of the resulting composition compared with a method not comprising a step of anion exchange chromatography or a method comprising instead a step of cation exchange chromatography.

[0235] The present invention provides a method for producing a composition comprising a multiplicity of AAV particles, wherein the method comprises a step of performing anion exchange chromatography on a sample comprising the AAV particle. In some embodiments, the step of anion exchange chromatography comprises passing the sample through an anion exchange column comprising quaternary polyethyleneimine.

[0236] In some embodiments, the AAV particle is an AAV particle according to the invention. The present invention also provides a composition comprising an AAV particle obtainable by or obtained by the method. In some embodiments, the composition has:

[0237] (a) a percentage of viral particles containing within their respective capsids the recombinant genome comprising the transgene that is greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90%;

[0238] (b) a percentage of viral particles comprising full capsids that is greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90%; and / or

[0239] (c) a full: empty capsid ratio that is greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85% or greater than 90%.

[0240] In some embodiments, the composition has a full:empty capsid ratio that is greater than 70%.

[0241] In some embodiments, the composition has:

[0242] 38 (a) a percentage of viral particles not containing within their respective capsids the recombinant genome comprising the transgene that is less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10%;

[0243] (b) a percentage of viral particles not comprising full capsids that is less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10%; and / or

[0244] (c) an empty:full capsid ratio that is less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10%.

[0245] In some embodiments, the composition has an empty:full capsid ratio that is less than 30%.

[0246] Vials

[0247] The present invention also provides a vial comprising a stopper and a composition of the invention. In some embodiments the stopper is a bromo-butyl or a chloro-butyl stopper.

[0248] 39 Informal Sequence Listing

[0249] SEQ Description Sequence

[0250] ID NO

[0251] 1 hGRKl GGGCCCCAGAAGCCTGGTGGTTGTTTGTCCTTCTCAGGGGAAAAGTGAG promoter GCGGCCCCTTGGAGGAAGGGGCCGGGCAGAATGATCTAATCGGATTCC AAGCAGCTCAGGGGATTGTCTTTTTCTAGCACCTTCTTGCCACTCCTAAG CGTCCTCCGTGACCCCGGCTGGGATTTAGCCTGGTGCTGTGTCAGCCCC GGGCTCCCAGGGGCTTCCCAGTGGTCCCCAGGAACCCTCGACAGGGCC AGGGCGTCTCTCTCGTCCAGCAAGGGCAGGGACGGGCCACAGGCCAAG GGC

[0252] 2 RPGR ATGAGAGAGCCAGAGGAGCTGATGCCAGATAGCGGAGCAGTGTTTACC transgene TTCGGAAAGTCCAAGTTCGCAGAGAATAACCCAGGAAAGTTCTGGTTTA AAAACGACGTGCCCGTCCACCTGTCTTGTGGCGATGAGCATAGTGCCGT GGTCACTGGGAACAATAAGCTGTATATGTTCGGGTCCAACAATTGGGGA CAGCTGGGGCTGGGATCCAAATCTGCTATCTCTAAGCCAACCTGCGTGA AGGCACTGAAACCCGAGAAGGTCAAACTGGCCGCTTGTGGCAGAAACC ACACTCTGGTGAGCACCGAGGGCGGGAATGTCTATGCCACCGGAGGCA ACAATGAGGGACAGCTGGGACTGGGGGACACTGAGGAAAGGAATACCT TTCACGTGATCTCCTTCTTTACATCTGAGCATAAGATCAAGCAGCTGAG CGCCGGCTCCAACACATCTGCAGCCCTGACTGAGGACGGGCGCCTGTTC ATGTGGGGAGATAATTCAGAGGGCCAGATTGGGCTGAAAAACGTGAGC AACGTGTGCGTGCCTCAGCAGGTGACCATCGGAAAGCCAGTCAGTTGG ATTTCATGTGGCTACTATCATAGCGCCTTCGTGACCACAGATGGCGAGC TGTACGTCTTTGGGGAGCCCGAAAACGGAAAACTGGGCCTGCCTAACC AGCTGCTGGGCAATCACCGGACACCCCAGCTGGTGTCCGAGATCCCTGA AAAAGTGATCCAGGTCGCCTGCGGGGGAGAGCATACAGTGGTCCTGAC TGAGAATGCCGTGTACACCTTCGGACTGGGCCAGTTTGGCCAGCTGGGG CTGGGAACCTTCCTGTTTGAGACATCCGAACCAAAAGTGATCGAGAACA TTCGCGACCAGACTATCAGCTACATTTCCTGCGGAGAGAATCACACCGC ACTGATCACAGACATTGGCCTGATGTATACCTTTGGCGATGGGCGGCAC GGGAAGCTGGGACTGGGCCTGGAGAACTTCACTAATCACTTCATCCCCA CCCTGTGCTCTAACTTCCTGCGGTTCATCGTGAAACTGGTCGCTTGCGGC GGGTGTCACATGGTGGTCTTCGCTGCACCTCATAGGGGCGTGGCTAAGG AGATCGAATTTGACGAGATTAACGATACATGCCTGAGCGTGGCAACTTT CCTGCCATACAGCTCCCTGACTTCTGGCAATGTGCTGCAGAGAACCCTG AGTGCAAGGATGCGGAGAAGGGAGAGGGAACGCTCTCCTGACAGTTTC TCAATGCGACGAACCCTGCCACCTATCGAGGGGACACTGGGACTGAGT

[0253]

[0254] GCCTGCTTCCTGCCTAACTCAGTGTTTCCACGATGTAGCGAGCGGAATC TGCAGGAGTCTGTCCTGAGTGAGCAGGATCTGATGCAGCCAGAGGAAC CCGACTACCTGCTGGATGAGATGACCAAGGAGGCCGAAATCGACAACT CTAGTACAGTGGAGTCCCTGGGCGAGACTACCGATATCCTGAATATGAC ACACATTATGTCACTGAACAGCAATGAGAAGAGTCTGAAACTGTCACC AGTGCAGAAGCAGAAGAAACAGCAGACTATTGGCGAGCTGACTCAGGA CACCGCCCTGACAGAGAACGACGATAGCGATGAGTATGAGGAAATGTC CGAGATGAAGGAAGGCAAAGCTTGTAAGCAGCATGTGAGTCAGGGGAT CTTCATGACACAGCCAGCCACAACTATTGAGGCTTTTTCAGACGAGGAA GTGGAGATCCCCGAGGAAAAAGAGGGCGCAGAAGATTCCAAGGGGAA TGGAATTGAGGAACAGGAGGTGGAAGCCAACGAGGAAAATGTGAAAG TCCACGGAGGCAGGAAGGAGAAAACAGAAATCCTGTCTGACGATCTGA CTGACAAGGCCGAGGTGTCCGAAGGCAAGGCAAAATCTGTCGGAGAGG CAGAAGACGGACCAGAGGGACGAGGGGATGGAACCTGCGAGGAAGGC TCAAGCGGGGCTGAGCATTGGCAGGACGAGGAACGAGAGAAGGGCGA AAAGGATAAAGGCCGCGGGGAGATGGAACGACCTGGAGAGGGCGAAA AAGAGCTGGCAGAGAAGGAGGAATGGAAGAAAAGGGACGGCGAGGAA CAGGAGCAGAAAGAAAGGGAGCAGGGCCACCAGAAGGAGCGCAACCA GGAGATGGAAGAGGGCGGCGAGGAAGAGCATGGCGAGGGAGAAGAGG AAGAGGGCGATAGAGAAGAGGAAGAGGAAAAAGAAGGCGAAGGGAA GGAGGAAGGAGAGGGCGAGGAAGTGGAAGGCGAGAGGGAAAAGGAG GAAGGAGAACGGAAGAAAGAGGAAAGAGCCGGCAAAGAGGAAAAGG GCGAGGAAGAGGGCGATCAGGGCGAAGGCGAGGAGGAAGAGACCGAG GGCCGCGGGGAAGAGAAAGAGGAGGGAGGAGAGGTGGAGGGCGGAGA GGTCGAAGAGGGAAAGGGCGAGCGCGAAGAGGAAGAGGAAGAGGGCG AGGGCGAGGAAGAAGAGGGCGAGGGGGAAGAAGAGGAGGGAGAGGG CGAAGAGGAAGAGGGGGAGGGAAAGGGCGAAGAGGAAGGAGAGGAA GGGGAGGGAGAGGAAGAGGGGGAGGAGGGCGAGGGGGAAGGCGAGG AGGAAGAAGGAGAGGGGGAAGGCGAAGAGGAAGGCGAGGGGGAAGG AGAGGAGGAAGAAGGGGAAGGCGAAGGCGAAGAGGAGGGAGAAGGA GAGGGGGAGGAAGAGGAAGGAGAAGGGAAGGGCGAGGAGGAAGGCG AAGAGGGAGAGGGGGAAGGCGAGGAAGAGGAAGGCGAGGGCGAAGG AGAGGACGGCGAGGGCGAGGGAGAAGAGGAGGAAGGGGAATGGGAA GGCGAAGAAGAGGAAGGCGAAGGCGAAGGCGAAGAAGAGGGCGAAG GGGAGGGCGAGGAGGGCGAAGGCGAAGGGGAGGAAGAGGAAGGCGA AGGAGAAGGCGAGGAAGAAGAGGGAGAGGAGGAAGGCGAGGAGGAA GGAGAGGGGGAGGAGGAGGGAGAAGGCGAGGGCGAAGAAGAAGAAG AGGGAGAAGTGGAGGGCGAAGTCGAGGGGGAGGAGGGAGAAGGGGAA

[0255]

[0256] 41 GGGGAGGAAGAAGAGGGCGAAGAAGAAGGCGAGGAAAGAGAAAAAG AGGGAGAAGGCGAGGAAAACCGGAGAAATAGGGAAGAGGAGGAAGA GGAAGAGGGAAAGTACCAGGAGACAGGCGAAGAGGAAAACGAGCGGC AGGATGGCGAGGAATATAAGAAAGTGAGCAAGATCAAAGGATCCGTCA AGTACGGCAAGCACAAAACCTATCAGAAGAAAAGCGTGACCAACACAC AGGGGAATGGAAAAGAGCAGCGAAGTAAAATGCCTGTGCAGTCAAAAC GGCTGCTGAAGAATGGCCCAAGCGGGTCTAAAAAATTCTGGAACAATG TCCTGCCACACTATCTGGAACTGAAGTAA

[0257] 5’ ITR GGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCA AAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAG CGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT

[0258] 3’ ITR AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTC GCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGC CCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCC SV40 late AACTGAAAAACCAGAAAGTTAACTGGTAAGTTTAGTCTTTTTGTCTTTT intron ATTTCAGGTCCCGGATCCGGTGGTGGTGCAAATCAAAGAACTGCTCCTC AGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTACTTCTGCT CTAAAAGCT SV40 late GGATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAAC polyA TAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATT GCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACA ATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTT TA RPGR ATGAGAGAGC CAGAGGAGCT GATGCCAGAC AGTGGAGCAG

[0259] transgene TGTTTACATT CGGAAAATCT AAGTTCGCTG AAAATAACCC AGGAAAGTTC TGGTTTAAAA ACGACGTGCC CGTCCACCTG TCTTGTGGCG ATGAGCATAG TGCCGTGGTC ACTGGGAACA ATAAGCTGTA CATGTTCGGG TCCAACAACT GGGGACAGCT GGGGCTGGGA TCCAAATCTG CTATCTCTAA GCCAACCTGC GTGAAGGCAC TGAAACCCGA GAAGGTCAAA CTGGCCGCTT GTGGCAGAAA CCACACTCTG GTGAGCACCG AGGGCGGGAA TGTCTATGCC ACCGGAGGCA ACAATGAGGG ACAGCTGGGA CTGGGGGACA CTGAGGAAAG GAATACCTTT CACGTGATCT CCTTCTTTAC ATCTGAGCAT AAGATCAAGC AGCTGAGCGC TGGCTCCAAC ACATCTGCAG CCCTGACTGA GGACGGGCGC CTGTTCATGT GGGGAGATAA TTCAGAGGGC CAGATTGGGC TGAAAAACGT GAGCAATGTG TGCGTCCCTC AGCAGGTGAC CATCGGAAAG CCAGTCAGTT GGATTTCATG TGGCTACTAT

[0260]

[0261] CATAGCGCCT TCGTGACCAC AGATGGCGAG CTGTACGTCT TTGGGGAGCC CGAAAACGGA AAACTGGGCC TGCCTAACCA GCTGCTGGGC AATCACCGGA CACCCCAGCT GGTGTCCGAG ATCCCTGAAA AAGTGATCCA GGTCGCCTGC GGGGGAGAGC ATACAGTGGT CCTGACTGAG AATGCTGTGT ATACCTTCGG ACTGGGCCAG TTTGGCCAGC TGGGGCTGGG AACCTTCCTG TTTGAGACAT CCGAACCAAA AGTGATCGAG AACATTCGCG ACCAGACTAT CAGCTACATT TCCTGCGGAG AGAATCACAC CGCACTGATC ACAGACATTG GCCTGATGTA TACCTTTGGC GATGGACGAC ACGGGAAGCT GGGACTGGGA CTGGAGAACT TCACTAATCA TTTTATCCCC ACCCTGTGTT CTAACTTCCT GCGGTTCATC GTGAAACTGG TCGCTTGCGG CGGGTGTCAC ATGGTGGTCT TCGCTGCACC TCATAGGGGC GTGGCTAAGG AGATCGAATT TGACGAGATT AACGATACAT GCCTGAGCGT GGCAACTTTC CTGCCATACA GCTCCCTGAC TTCTGGCAAT GTGCTGCAGA GAACCCTGAG TGCAAGGATG CGGAGAAGGG AGAGGGAACG CTCTCCTGAC AGTTTCTCAA TGCGACGAAC CCTGCCACCT ATCGAGGGAA CACTGGGACT GAGTGCCTGC TTCCTGCCTA ACTCAGTGTT TCCACGATGT AGCGAGCGGA ATCTGCAGGA GTCTGTCCTG AGTGAGCAGG ATCTGATGCA GCCAGAGGAA CCCGACTACC TGCTGGATGA GATGACCAAG GAGGCCGAAA TCGACAACTC TAGTACAGTG GAGTCCCTGG GCGAGACTAC CGATATCCTG AATATGACAC ACATTATGTC ACTGAACAGC AATGAGAAGA GTCTGAAACT GTCACCAGTG CAGAAGCAGA AGAAACAGCA GACTATTGGC GAGCTGACTC AGGACACCGC CCTGACAGAG AACGACGATA GCGATGAGTA TGAGGAAATG TCCGAGATGA AGGAAGGCAA AGCTTGTAAG CAGCATGTCA GTCAGGGGAT CTTCATGACA CAGCCAGCCA CAACTATTGA GGCTTTTTCA GACGAGGAAG TGGAGATCCC CGAGGAAAAA GAGGGCGCAG AAGATTCCAA GGGGAATGGA ATTGAGGAAC AGGAGGTGGA AGCCAACGAG GAAAATGTGA AAGTCCACGG AGGCAGGAAG GAGAAAACAG AAATCCTGTC TGACGATCTG ACTGACAAGG CCGAGGTGTC CGAAGGCAAG GCAAAATCTG TCGGAGAGGC AGAAGACGGA CCAGAGGGAC GAGGGGATGG AACCTGCGAG GAAGGCTCAA GCGGGGCTGA GCATTGGCAG GACGAGGAAC GAGAGAAGGG CGAAAAGGAT AAAGGCCGCG GGGAGATGGA ACGACCTGGA GAGGGCGAAA AAGAGCTGGC AGAGAAGGAG GAATGGAAGA AAAGGGACGG

[0262]

[0263] CGAGGAACAG GAGCAGAAAG AAAGGGAGCA GGGCCACCAG AAGGAGCGCA ACCAGGAGAT GGAAGAGGGC GGCGAGGAAG AGCATGGCGA GGGAGAAGAG GAAGAGGGCG ATAGAGAAGA GGAAGAGGAA AAAGAAGGCG AAGGGAAGGA GGAAGGAGAG GGCGAGGAAG TGGAAGGCGA GAGGGAAAAG GAGGAAGGAG AACGGAAGAA AGAGGAAAGA GCCGGCAAAG AGGAAAAGGG CGAGGAAGAG GGCGATCAGG GCGAAGGCGA GGAGGAAGAG ACCGAGGGCC GCGGGGAAGA GAAAGAGGAG GGAGGAGAGG TGGAGGGCGG AGAGGTCGAA GAGGGAAAGG GCGAGCGCGA AGAGGAAGAG GAAGAGGGCG AGGGCGAGGA AGAAGAGGGC GAGGGGGAAG AAGAGGAGGG AGAGGGCGAA GAGGAAGAGG GGGAGGGAAA GGGCGAAGAG GAAGGAGAGG AAGGGGAGGG AGAGGAAGAG GGGGAGGAGG GCGAGGGGGA AGGCGAGGAG GAAGAAGGAG AGGGGGAAGG CGAAGAGGAA GGCGAGGGGG AAGGAGAGGA GGAAGAAGGG GAAGGCGAAG GCGAAGAGGA GGGAGAAGGA GAGGGGGAGG AAGAGGAAGG AGAAGGGAAG GGCGAGGAGG AAGGCGAAGA GGGAGAGGGG GAAGGCGAGG AAGAGGAAGG CGAGGGCGAA GGAGAGGACG GCGAGGGCGA GGGAGAAGAG GAGGAAGGGG AATGGGAAGG CGAAGAAGAG GAAGGCGAAG GCGAAGGCGA AGAAGAGGGC GAAGGGGAGG GCGAGGAGGG CGAAGGCGAA GGGGAGGAAG AGGAAGGCGA AGGAGAAGGC GAGGAAGAAG AGGGAGAGGA GGAAGGCGAG GAGGAAGGAG AGGGGGAGGA GGAGGGAGAA GGCGAGGGCG AAGAAGAAGA AGAGGGAGAA GTGGAGGGCG AAGTCGAGGG GGAGGAGGGA GAAGGGGAAG GGGAGGAAGA AGAGGGCGAA GAAGAAGGCG AGGAAAGAGA AAAAGAGGGA GAAGGCGAGG AAAACCGGAG AAATAGGGAA GAGGAGGAAG AGGAAGAGGG AAAGTACCAG GAGACAGGCG AAGAGGAAAA CGAGCGGCAG GATGGCGAGG AATATAAGAA AGTGAGCAAG ATCAAAGGAT CCGTCAAGTA CGGCAAGCAC AAAACCTATC AGAAGAAAAG CGTGACCAAC ACACAGGGGA ATGGAAAAGA GCAGAGGAGT AAGATGCCTG TGCAGTCAAA ACGGCTGCTG AAGAATGGCC CATCTGGAAG TAAAAAATTC TGGAACAATG TGCTGCCCCA CTATCTGGAA CTGAAATAA

[0264] Vector GGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCA genome AAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAG sequence CGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTCA

[0265] GATCTGAATTCGGGCCCCAGAAGCCTGGTGGTTGTTTGTCCTTCTCAGG

[0266]

[0267] GGAAAAGTGAGGCGGCCCCTTGGAGGAAGGGGCCGGGCAGAATGATCT AATCGGATTCCAAGCAGCTCAGGGGATTGTCTTTTTCTAGCACCTTCTTG CCACTCCTAAGCGTCCTCCGTGACCCCGGCTGGGATTTAGCCTGGTGCT GTGTCAGCCCCGGGCTCCCAGGGGCTTCCCAGTGGTCCCCAGGAACCCT CGACAGGGCCAGGGCGTCTCTCTCGTCCAGCAAGGGCAGGGACGGGCC ACAGGCCAAGGGCTCTAGACTCGAGGAACTGAAAAACCAGAAAGTTAA CTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCGGATCCGGTGG TGGTGCAAATCAAAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTA GGCCTGTACGGAAGTGTTACTTCTGCTCTAAAAGCTGCGGAATTGTACC CGCGGCCGCGCCACCATGAGAGAGCCAGAGGAGCTGATGCCAGATAGC GGAGCAGTGTTTACCTTCGGAAAGTCCAAGTTCGCAGAGAATAACCCA GGAAAGTTCTGGTTTAAAAACGACGTGCCCGTCCACCTGTCTTGTGGCG ATGAGCATAGTGCCGTGGTCACTGGGAACAATAAGCTGTATATGTTCGG GTCCAACAATTGGGGACAGCTGGGGCTGGGATCCAAATCTGCTATCTCT AAGCCAACCTGCGTGAAGGCACTGAAACCCGAGAAGGTCAAACTGGCC GCTTGTGGCAGAAACCACACTCTGGTGAGCACCGAGGGCGGGAATGTC TATGCCACCGGAGGCAACAATGAGGGACAGCTGGGACTGGGGGACACT GAGGAAAGGAATACCTTTCACGTGATCTCCTTCTTTACATCTGAGCATA AGATCAAGCAGCTGAGCGCCGGCTCCAACACATCTGCAGCCCTGACTG AGGACGGGCGCCTGTTCATGTGGGGAGATAATTCAGAGGGCCAGATTG GGCTGAAAAACGTGAGCAACGTGTGCGTGCCTCAGCAGGTGACCATCG GAAAGCCAGTCAGTTGGATTTCATGTGGCTACTATCATAGCGCCTTCGT GACCACAGATGGCGAGCTGTACGTCTTTGGGGAGCCCGAAAACGGAAA ACTGGGCCTGCCTAACCAGCTGCTGGGCAATCACCGGACACCCCAGCTG GTGTCCGAGATCCCTGAAAAAGTGATCCAGGTCGCCTGCGGGGGAGAG CATACAGTGGTCCTGACTGAGAATGCCGTGTACACCTTCGGACTGGGCC AGTTTGGCCAGCTGGGGCTGGGAACCTTCCTGTTTGAGACATCCGAACC AAAAGTGATCGAGAACATTCGCGACCAGACTATCAGCTACATTTCCTGC GGAGAGAATCACACCGCACTGATCACAGACATTGGCCTGATGTATACCT TTGGCGATGGGCGGCACGGGAAGCTGGGACTGGGCCTGGAGAACTTCA CTAATCACTTCATCCCCACCCTGTGCTCTAACTTCCTGCGGTTCATCGTG AAACTGGTCGCTTGCGGCGGGTGTCACATGGTGGTCTTCGCTGCACCTC ATAGGGGCGTGGCTAAGGAGATCGAATTTGACGAGATTAACGATACAT GCCTGAGCGTGGCAACTTTCCTGCCATACAGCTCCCTGACTTCTGGCAA TGTGCTGCAGAGAACCCTGAGTGCAAGGATGCGGAGAAGGGAGAGGGA ACGCTCTCCTGACAGTTTCTCAATGCGACGAACCCTGCCACCTATCGAG GGGACACTGGGACTGAGTGCCTGCTTCCTGCCTAACTCAGTGTTTCCAC GATGTAGCGAGCGGAATCTGCAGGAGTCTGTCCTGAGTGAGCAGGATC

[0268]

[0269] 45 TGATGCAGCCAGAGGAACCCGACTACCTGCTGGATGAGATGACCAAGG AGGCCGAAATCGACAACTCTAGTACAGTGGAGTCCCTGGGCGAGACTA CCGATATCCTGAATATGACACACATTATGTCACTGAACAGCAATGAGAA GAGTCTGAAACTGTCACCAGTGCAGAAGCAGAAGAAACAGCAGACTAT TGGCGAGCTGACTCAGGACACCGCCCTGACAGAGAACGACGATAGCGA TGAGTATGAGGAAATGTCCGAGATGAAGGAAGGCAAAGCTTGTAAGCA GCATGTGAGTCAGGGGATCTTCATGACACAGCCAGCCACAACTATTGAG GCTTTTTCAGACGAGGAAGTGGAGATCCCCGAGGAAAAAGAGGGCGCA GAAGATTCCAAGGGGAATGGAATTGAGGAACAGGAGGTGGAAGCCAA CGAGGAAAATGTGAAAGTCCACGGAGGCAGGAAGGAGAAAACAGAAA TCCTGTCTGACGATCTGACTGACAAGGCCGAGGTGTCCGAAGGCAAGG CAAAATCTGTCGGAGAGGCAGAAGACGGACCAGAGGGACGAGGGGAT GGAACCTGCGAGGAAGGCTCAAGCGGGGCTGAGCATTGGCAGGACGAG GAACGAGAGAAGGGCGAAAAGGATAAAGGCCGCGGGGAGATGGAACG ACCTGGAGAGGGCGAAAAAGAGCTGGCAGAGAAGGAGGAATGGAAGA AAAGGGACGGCGAGGAACAGGAGCAGAAAGAAAGGGAGCAGGGCCAC CAGAAGGAGCGCAACCAGGAGATGGAAGAGGGCGGCGAGGAAGAGCA TGGCGAGGGAGAAGAGGAAGAGGGCGATAGAGAAGAGGAAGAGGAAA AAGAAGGCGAAGGGAAGGAGGAAGGAGAGGGCGAGGAAGTGGAAGG CGAGAGGGAAAAGGAGGAAGGAGAACGGAAGAAAGAGGAAAGAGCC GGCAAAGAGGAAAAGGGCGAGGAAGAGGGCGATCAGGGCGAAGGCGA GGAGGAAGAGACCGAGGGCCGCGGGGAAGAGAAAGAGGAGGGAGGA GAGGTGGAGGGCGGAGAGGTCGAAGAGGGAAAGGGCGAGCGCGAAGA GGAAGAGGAAGAGGGCGAGGGCGAGGAAGAAGAGGGCGAGGGGGAA GAAGAGGAGGGAGAGGGCGAAGAGGAAGAGGGGGAGGGAAAGGGCG AAGAGGAAGGAGAGGAAGGGGAGGGAGAGGAAGAGGGGGAGGAGGG CGAGGGGGAAGGCGAGGAGGAAGAAGGAGAGGGGGAAGGCGAAGAG GAAGGCGAGGGGGAAGGAGAGGAGGAAGAAGGGGAAGGCGAAGGCG AAGAGGAGGGAGAAGGAGAGGGGGAGGAAGAGGAAGGAGAAGGGAA GGGCGAGGAGGAAGGCGAAGAGGGAGAGGGGGAAGGCGAGGAAGAG GAAGGCGAGGGCGAAGGAGAGGACGGCGAGGGCGAGGGAGAAGAGG AGGAAGGGGAATGGGAAGGCGAAGAAGAGGAAGGCGAAGGCGAAGGC GAAGAAGAGGGCGAAGGGGAGGGCGAGGAGGGCGAAGGCGAAGGGG AGGAAGAGGAAGGCGAAGGAGAAGGCGAGGAAGAAGAGGGAGAGGA GGAAGGCGAGGAGGAAGGAGAGGGGGAGGAGGAGGGAGAAGGCGAG GGCGAAGAAGAAGAAGAGGGAGAAGTGGAGGGCGAAGTCGAGGGGGA GGAGGGAGAAGGGGAAGGGGAGGAAGAAGAGGGCGAAGAAGAAGGC GAGGAAAGAGAAAAAGAGGGAGAAGGCGAGGAAAACCGGAGAAATA

[0270]

[0271] 46 GGGAAGAGGAGGAAGAGGAAGAGGGAAAGTACCAGGAGACAGGCGAA GAGGAAAACGAGCGGCAGGATGGCGAGGAATATAAGAAAGTGAGCAA GATCAAAGGATCCGTCAAGTACGGCAAGCACAAAACCTATCAGAAGAA AAGCGTGACCAACACACAGGGGAATGGAAAAGAGCAGCGAAGTAAAA TGCCTGTGCAGTCAAAACGGCTGCTGAAGAATGGCCCAAGCGGGTCTA AAAAATTCTGGAACAATGTCCTGCCACACTATCTGGAACTGAAGTAAGC GGCCGCGCGGATCCAGACATGATAAGATACATTGATGAGTTTGGACAA ACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTG ATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAA CAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGG GAGGTTTTTTAGCATGCTGGGGAGAGATCTGAGGAACCCCTAGTGATGG AGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCG ACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG CGAGCGAGCGCGCAGAGAGGGAGTGGCC AAV2- MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGY derived KYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAE capsid FQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSP VEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGT NTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWA LPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQR LINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQ LPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPS QMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYFLSRTNTPS GTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEFSW TGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNV DIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPG MVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVP ANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNK SVNVDFTVDTNGVYSEPRPIGTRFLTRNL

[0272] hGRK1 GGGCCCCAGAAGCCTGGTGGTTGTTTGTCCTTCTCAGGGGAAAAGTGAG promoter GCG GCCCCTTGGAGGAAGGGGCCGGGCAGAATGAT CTAATCGGATTCCAAGCAGCTCAGGGGATTGTCTTTTTCTAGCACCTTCT TGCCACTCCTAAGCGTCCTCCGTGACCCCGGCTGGGATTTAGCCTGGTG CTGTGTCAGCCCCGGGCTCCCAGGGGCTTCCCAGTGGTCCCCAGGAACC CTCGACAGGGCCCGGTCTCTCTCGTCCA GCAAGGGCAGGGACGGGCCACAGGCCAAGGGC

[0273]

[0274] RPGR MREPEELMPDSGAVFTFGKSKFAENNPGKFWFKNDVPVHLSCGDEHSAVV ORF 15 TGNNKLYMFGSNNWGQLGLGSKSAISKPTCVKALKPEKVKLAACGRNHT polypeptide LVSTEGGNVYATGGNNEGQLGLGDTEERNTFHVISFFTSEHKIKQLSAGSN TSAALTEDGRLFMWGDNSEGQIGLKNVSNVCVPQQVTIGKPVSWISCGYY HSAFVTTDGELYVFGEPENGKLGLPNQLLGNHRTPQLVSEIPEKVIQVACG GEHTVVLTENAVYTFGLGQFGQLGLGTFLFETSEPKVIENIRDQTISYISCGE NHTALITDIGLMYTFGDGRHGKLGLGLENFTNHFIPTLCSNFLRFIVKLVAC GGCHMVVFAAPHRGVAKEIEFDEINDTCLSVATFLPYSSLTSGNVLQRTLS ARMRRRERERSPDSFSMRRTLPPIEGTLGLSACFLPNSVFPRCSERNLQESV LSEQDLMQPEEPDYLLDEMTKEAEIDNSSTVESLGETTDILNMTHIMSLNSN EKSLKLSPVQKQKKQQTIGELTQDTALTENDDSDEYEEMSEMKEGKACKQ HVSQGIFMTQPATTIEAFSDEEVEIPEEKEGAEDSKGNGIEEQEVEANEENV KVHGGRKEKTEILSDDLTDKAEVSEGKAKSVGEAEDGPEGRGDGTCEEGS SGAEHWQDEEREKGEKDKGRGEMERPGEGEKELAEKEEWKKRDGEEQEQ KEREQGHQKERNQEMEEGGEEEHGEGEEEEGDREEEEEKEGEGKEEGEGE EVEGEREKEEGERKKEERAGKEEKGEEEGDQGEGEEEETEGRGEEKEEGG EVEGGEVEEGKGEREEEEEEGEGEEEEGEGEEEEGEGEEEEGEGKGEEEGE EGEGEEEGEEGEGEGEEEEGEGEGEEEGEGEGEEEEGEGEGEEEGEGEGEE EEGEGKGEEEGEEGEGEGEEEEGEGEGEDGEGEGEEEEGEWEGEEEEGEGE GEEEGEGEGEEGEGEGEEEEGEGEGEEEEGEEEGEEEGEGEEEGEGEGEEE EEGEVEGEVEGEEGEGEGEEEEGEEEGEEREKEGEGEENRRNREEEEEEEG KYQETGEEENERQDGEEYKKVSKIKGSVKYGKHKTYQKKSVTNTQGNGK EQRSKMPVQSKRLLKNGPSGSKKFWNNVLPHYLELK SV40 late GTAAGTTTAG TCTTTTTGTC TTTTATTTCA GGTCCCGGAT CCGGTGGTGG TGCAAATCAA AGAACTGCTC CTCAGTGGAT

[0275] intron

[0276] GTTGCCTTTA CTTCTAG SV40 late GATCCAGACA TGATAAGATA CATTGATGAG TTTGGACAAA CCACAACTAG AATGCAGTGA AAAAAATGCT TTATTTGTGA

[0277] polyA

[0278] AATTTGTGAT GCTATTGCTT TATTTGTAAC CATTATAAGC TGCAATAAAC AAGTT AAV5 MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYN YLGPGNGLDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQ

[0279] capsid

[0280] EKLADDTSFGGNLGKAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPK

[0281] sequence RKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPL GDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYR EIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFR PRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGT EGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNN FEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKN LAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGAS YQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSE SETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDV

[0282]

[0283] YLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDV PVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAP DSTGEYRTTRPIGTRYLTRPL

[0284] Alternative ATGAGAGAGCCCGAGGAACTGATGCCCGATAGCGGAGCCGTCTTCACC TTTGGGAAATCTAAATTCGCAGAGAACAACCCTGGAAAATTCTGGTTTA RPGR AGAACGACGTGCCCGTGCACCTGAGCTGTGGCGATGAGCACTCCGCCGT

[0285] sequence GGTGACAGGCAACAATAAGCTGTACATGTTCGGCTCTAACAATTGGGG ACAGCTGGGCCTGGGAAGCAAGTCCGCCATCAGCAAGCCAACCTGCGT GAAGGCCCTGAAGCCCGAGAAGGTGAAGCTGGCCGCCTGTGGCAGAAA CCACACACTGGTGAGCACCGAGGGCGGCAATGTGTATGCCACAGGCGG CAACAATGAAGGACAGCTGGGCCTGGGCGACACAGAGGAGAGGAATA CCTTTCACGTGATCAGCTTCTTTACCTCCGAGCACAAGATCAAGCAGCT GTCCGCCGGCTCTAACACATCTGCAGCACTGACAGAGGATGGAAGACT GTTCATGTGGGGCGATAATAGCGAGGGCCAGATCGGCCTGAAGAACGT GTCCAATGTGTGCGTGCCTCAGCAGGTGACCATCGGCAAGCCAGTGTCC TGGATCTCTTGTGGCTACTATCACAGCGCCTTCGTGACCACAGATGGCG AGCTGTACGTGTTTGGAGAGCCTGAGAATGGCAAGCTGGGCCTGCCTAA CCAGCTGCTGGGCAATCACCGGACACCCCAGCTGGTGTCCGAGATCCCT GAGAAAGTGATCCAGGTGGCATGTGGCGGCGAGCACACAGTGGTGCTG ACCGAGAATGCCGTGTATACCTTTGGCCTGGGACAGTTTGGCCAGCTGG GCCTGGGCACATTCCTGTTTGAGACATCCGAGCCAAAAGTGATCGAGAA CATCCGCGACCAGACAATCAGCTACATCTCCTGCGGCGAGAATCACACA GCCCTGATCACCGACATCGGCCTGATGTATACCTTTGGCGATGGAAGAC ACGGCAAGCTGGGCCTGGGCCTGGAGAACTTCACAAATCACTTTATCCC CACCCTGTGTTCTAACTTCCTGCGGTTCATCGTGAAGCTGGTGGCCTGCG GCGGATGTCACATGGTGGTGTTTGCAGCCCCTCACAGGGGCGTGGCCAA GGAGATCGAGTTTGACGAGATCAACGATACATGCCTGTCCGTGGCCACC TTCCTGCCATACAGCTCCCTGACATCCGGCAATGTGCTGCAGAGAACCC TGTCTGCAAGAATGAGAAGAAGGGAGAGAGAGCGGTCCCCTGACTCTT TCAGCATGAGAAGAACACTGCCCCCTATTGAGGGAACCCTGGGCCTGTC TGCCTGCTTCCTGCCTAACTCTGTGTTTCCAAGATGTAGCGAGAGGAAT CTGCAGGAGTCTGTGCTGAGCGAGCAGGATCTGATGCAGCCAGAGGAG CCCGACTACCTGCTGGATGAGATGACAAAGGAGGCCGAGATCGACAAC TCTAGCACCGTGGAGAGCCTGGGCGAGACAACAGATATCCTGAATATG ACACACATCATGTCCCTGAACTCTAATGAGAAGTCTCTGAAGCTGAGCC CAGTGCAGAAGCAGAAGAAGCAGCAGACCATCGGCGAGCTGACCCAGG ACACAGCCCTGACCGAGAACGACGATTCTGATGAGTATGAGGAGATGA GCGAGATGAAGGAGGGCAAGGCCTGTAAGCAGCACGTGTCCCAGGGCA TCTTCATGACCCAGCCAGCCACCACAATCGAGGCCTTTTCTGACGAGGA GGTGGAGATCCCCGAGGAGAAGGAGGGCGCCGAGGATAGCAAGGGCA ATGGCATCGAGGAGCAGGAGGTGGAGGCCAACGAGGAGAATGTGAAG GTGCACGGCGGAAGAAAGGAGAAGACAGAGATCCTGTCCGACGATCTG ACCGACAAGGCCGAGGTGTCCGAGGGCAAGGCCAAGTCTGTGGGAGAG GCAGAGGATGGACCTGAGGGACGCGGCGATGGAACATGTGAGGAGGG CTCCTCTGGAGCAGAGCACTGGCAGGATGAGGAGAGAGAGAAGGGCGA GAAGGATAAGGGCAGAGGCGAGATGGAGAGGCCTGGAGAGGGAGAGA AGGAGCTGGCAGAGAAGGAGGAGTGGAAGAAGAGGGATGGCGAGGAG CAGGAGCAGAAGGAGAGAGAGCAGGGCCACCAGAAAGAGAGGAACCA GGAGATGGAAGAGGGCGGCGAGGAGGAGCACGGAGAGGGAGAGGAGG AGGAGGGCGATAGAGAAGAAGAGGAGGAGAAAGAGGGAGAGGGCAA GGAGGAGGGAGAGGGAGAAGAAGTGGAAGGAGAGAGAGAGAAGGAG GAAGGAGAGCGCAAGAAGGAAGAAAGAGCAGGCAAGGAGGAGAAAG GAGAGGAGGAGGGCGATCAGGGAGAAGGAGAGGAGGAGGAGACAGA AGGACGCGGCGAGGAAAAAGAGGAGGGCGGCGAGGTCGAGGGCGGCG AGGTCGAAGAGGGCAAGGGCGAAAGAGAAGAAGAGGAGGAGGAAGGC GAGGGCGAAGAAGAGGAGGGCGAGGGCGAGGAAGAAGAGGGCGAGG GCGAAGAGGAAGAAGGAGAGGGCAAGGGCGAGGAGGAGGGCGAAGA

[0286]

[0287] AGGCGAAGGGGAGGAGGAGGGCGAAGAGGGAGAGGGCGAGGGCGAG GAGGAAGAAGGCGAAGGAGAAGGCGAAGAAGAAGGAGAAGGAGAGG GAGAAGAGGAGGAAGGCGAAGGAGAGGGGGAAGAGGAAGGAGAAGG GGAGGGCGAAGAGGAGGAGGGAGAAGGCAAGGGAGAGGAGGAGGGC GAGGAAGGAGAAGGCGAAGGCGAGGAGGAGGAAGGAGAGGGAGAAG GAGAAGATGGAGAAGGAGAGGGCGAGGAAGAGGAAGGAGAGTGGGA GGGCGAGGAAGAGGAGGGAGAAGGAGAAGGAGAAGAAGAAGGAGAA GGCGAGGGAGAAGAAGGAGAGGGAGAAGGGGAAGAAGAGGAGGGGG AAGGAGAGGGCGAGGAGGAAGAGGGAGAAGAAGAAGGCGAAGAAGA GGGAGAAGGCGAGGAAGAAGGAGAGGGAGAGGGGGAAGAGGAGGAA GAGGGCGAGGTGGAAGGAGAGGTGGAGGGCGAAGAGGGGGAAGGGGA AGGAGAAGAAGAAGAAGGAGAGGAGGAGGGAGAGGAGAGAGAGAAA GAAGGCGAGGGCGAGGAGAACAGAAGGAATCGCGAAGAAGAAGAAGA AGAGGAGGGCAAGTACCAGGAGACAGGCGAGGAGGAGAACGAGCGGC AGGATGGCGAGGAGTATAAGAAGGTGTCCAAGATCAAGGGCTCTGTGA AGTACGGCAAGCACAAGACCTATCAGAAGAAGAGCGTGACCAACACAC AGGGCAATGGCAAGGAGCAGCGCAGCAAGATGCCTGTGCAGTCCAAGC GGCTGCTGAAGAATGGCCCAAGCGGGTCTAAAAAATTCTGGAACAATG TCCTGCCACACTATCTGGAACTGAAATAA

[0288] Alternative ATGAGAGAACCCGAGGAACTGATGCCTGACTCTGGCGCCGTGTTCACCT RPGR TCGGCAAGAGCAAGTTCGCCGAGAACAACCCCGGCAAGTTCTGGTTCA AGAACGACGTGCCAGTGCACCTGAGCTGTGGCGACGAACATTCTGCCGT

[0289] sequence GGTCACCGGCAACAACAAGCTGTACATGTTCGGCAGCAACAACTGGGG CCAGCTCGGCCTGGGATCTAAGAGCGCCATCAGCAAGCCTACCTGCGTG AAGGCCCTGAAGCCTGAGAAAGTGAAGCTGGCCGCCTGCGGCAGAAAT CACACCCTGGTTTCTACCGAAGGCGGCAACGTGTACGCCACCGGCGGA AACAATGAAGGACAGCTTGGACTGGGCGACACCGAGGAAAGAAACACC TTCCACGTGATCAGCTTTTTCACCAGCGAGCACAAGATCAAGCAGCTGA GCGCCGGCAGCAATACCTCTGCTGCCCTGACAGAAGATGGCCGGCTGTT CATGTGGGGCGACAATTCTGAGGGCCAGATCGGACTGAAGAACGTGTC CAATGTGTGCGTGCCCCAGCAAGTGACAATCGGCAAGCCTGTGTCCTGG ATCAGCTGCGGCTACTACCACAGCGCCTTCGTGACAACAGACGGCGAG CTGTATGTGTTCGGCGAGCCCGAGAATGGCAAGCTGGGACTGCCTAATC AGCTGCTGGGCAACCACAGAACCCCTCAGCTGGTGTCTGAGATCCCCGA AAAAGTGATCCAGGTGGCCTGTGGCGGAGAGCACACAGTGGTGCTGAC AGAGAATGCCGTGTACACATTTGGCCTGGGCCAGTTTGGCCAACTCGGA CTGGGCACCTTCCTGTTCGAGACAAGCGAGCCCAAAGTGATCGAGAAC ATCCGGGACCAGACCATCAGCTACATCTCTTGCGGCGAGAACCACACA GCCCTGATCACAGACATCGGCCTGATGTATACCTTCGGCGACGGCAGAC ACGGCAAACTCGGCCTTGGCCTGGAAAACTTCACCAACCACTTCATCCC TACTCTGTGCAGCAACTTCCTGCGGTTCATCGTGAAACTGGTGGCCTGC GGAGGCTGCCACATGGTGGTTTTTGCCGCTCCTCATAGAGGCGTGGCCA AAGAGATTGAGTTCGACGAGATCAACGATACCTGCCTGAGCGTGGCCA CCTTTCTGCCTTACAGCTCTCTGACCAGCGGCAATGTGCTGCAGAGGAC ACTGAGCGCCAGAATGCGCAGACGGGAAAGAGAGAGAAGCCCCGACA GCTTCAGCATGCGGAGAACCCTGCCTCCAATCGAGGGAACACTGGGCCT GAGCGCCTGCTTCCTGCCTAATAGCGTGTTCCCCAGATGCAGCGAGCGG AACCTGCAAGAGTCTGTGCTGAGCGAGCAGGACCTGATGCAGCCTGAG GAACCCGACTACCTGCTGGACGAGATGACCAAAGAGGCCGAGATCGAC AACAGCAGCACCGTGGAATCTCTGGGCGAGACAACCGACATCCTGAAC ATGACCCACATCATGAGCCTGAACAGCAACGAGAAGTCTCTGAAGCTG AGCCCCGTGCAGAAGCAGAAGAAGCAGCAGACCATCGGAGAGCTGACC CAGGATACCGCTCTGACCGAGAACGACGACAGCGACGAGTACGAAGAG ATGAGCGAGATGAAGGAAGGCAAGGCCTGCAAGCAGCACGTGTCCCAG GGCATCTTTATGACCCAGCCTGCCACCACCATCGAGGCCTTTTCCGACG AGGAAGTGGAAATCCCCGAGGAAAAAGAGGGCGCCGAGGACAGCAAA GGCAACGGCATTGAGGAACAAGAGGTGGAAGCCAACGAAGAGAACGT

[0290]

[0291] GAAGGTGCACGGCGGACGGAAAGAGAAAACAGAGATCCTGAGCGACG

[0292] 50 ACCTGACCGACAAGGCCGAAGTGTCTGAGGGCAAAGCCAAGTCTGTGG GCGAAGCCGAGGACGGCCCAGAAGGCAGAGGCGACGGAACATGTGAA GAGGGATCTAGCGGAGCCGAGCACTGGCAGGACGAAGAGAGAGAGAA AGGCGAGAAGGACAAAGGCAGGGGCGAGATGGAAAGACCTGGCGAGG GCGAAAAAGAGCTGGCCGAGAAAGAGGAGTGGAAGAAACGCGACGGC GAAGAACAAGAGCAGAAAGAACGCGAGCAGGGCCACCAGAAAGAAAG AAATCAAGAGATGGAAGAAGGCGGCGAGGAAGAACACGGCGAAGGGG AAGAAGAGGAAGGCGACCGAGAGGAAGAAGAAGAAAAAGAAGGCGA AGGCAAAGAGGAAGGCGAGGGCGAAGAGGTGGAAGGCGAGCGGGAAA AAGAAGAGGGCGAGCGCAAGAAAGAAGAAAGAGCCGGCAAAGAAGAG AAGGGCGAAGAAGAAGGCGATCAAGGCGAAGGCGAGGAAGAAGAAAC CGAAGGCCGCGGAGAAGAGAAAGAGGAAGGCGGCGAAGTTGAAGGCG GCGAGGTGGAAGAAGGCAAGGGCGAGAGAGAAGAAGAGGAAGAGGA AGGCGAAGGGGAAGAAGAAGAAGGCGAGGGCGAAGAGGAAGAAGGC GAAGGCGAGGAAGAGGAAGGCGAAGGCAAGGGCGAAGAGGAAGGCG AAGAAGGCGAGGGCGAAGAAGAGGGAGAAGAAGGCGAAGGCGAGGG CGAAGAAGAGGAAGGCGAAGGCGAAGGCGAGGAAGAAGGCGAAGGCG AAGGCGAAGAGGAAGAAGGCGAAGGCGAAGGGGAAGAAGAAGGCGA AGGCGAAGGCGAGGAAGAGGAAGGCGAAGGCAAAGGGGAAGAAGAG GGCGAAGAAGGCGAAGGCGAAGGCGAGGAAGAAGAAGGCGAAGGCGA AGGCGAAGACGGCGAAGGCGAGGGCGAAGAGGAAGAGGGCGAGTGGG AGGGCGAAGAAGAAGAAGGCGAAGGCGAGGGCGAAGAGGAAGGCGA AGGCGAAGGCGAAGAAGGCGAGGGCGAAGGCGAAGAAGAAGAAGGCG AAGGCGAAGGCGAAGAAGAGGAAGGGGAAGAAGAAGGCGAAGAGGA AGGCGAGGGCGAAGAAGAAGGCGAAGGCGAGGGCGAAGAAGAGGAA GAGGGCGAAGTTGAAGGGGAAGTTGAGGGCGAAGAAGGCGAAGGCGA AGGGGAAGAAGAGGAAGGCGAGGAAGAGGGCGAAGAACGCGAGAAA GAAGGCGAAGGGGAAGAGAACCGCCGGAACAGAGAAGAGGAAGAAGA AGAGGAAGGCAAGTACCAAGAGACAGGCGAGGAAGAGAACGAGCGGC AGGATGGCGAAGAGTACAAGAAGGTGTCCAAGATCAAGGGCAGCGTGA AGTACGGCAAGCACAAGACCTACCAGAAAAAGTCCGTGACCAACACAC AAGGCAATGGCAAAGAACAGCGGAGCAAGATGCCCGTGCAGTCCAAGA GACTGCTGAAGAATGGCCCCAGCGGCAGCAAAAAGTTCTGGAACAACG TGCTGCCCCACTACCTGGAACTGAAGTGA

[0293] Alternative GGGCCCCAGA AGCCTGGTGG TTGTTTGTCC TTCTCAGGGG

[0294] GRK1 AAAAGTGAGG CGGCCCCTTG GAGGAAGGGG CCGGGCAGAA TGATCTAATC GGATTCCAAG CAGCTCAGGG GATTGTCTTT

[0295] promoter TTCTAGCACC TTCTTGCCAC TCCTAAGCGT CCTCCGTGAC CCCGGCTGGG ATTTAGCCTG GTGCTGTGTC AGCCCCGGG RPGR ATGAGAGAGCCAGAGGAGCTGATGCCAGATAGCGGAGCAGTGTTTACC

[0296] transgene TTCGGAAAGTCCAAGTTCGCAGAGAATAACCCAGGAAAGTTCTGGTTTA AAAACGACGTGCCCGTCCACCTGTCTTGTGGCGATGAGCATAGTGCCGT GGTCACTGGGAACAATAAGCTGTATATGTTCGGGTCCAACAATTGGGGA CAGCTGGGGCTGGGATCCAAATCTGCTATCTCTAAGCCAACCTGCGTGA AGGCACTGAAACCCGAGAAGGTCAAACTGGCCGCTTGTGGCAGAAACC ACACTCTGGTGAGCACCGAGGGCGGGAATGTCTATGCCACCGGAGGCA ACAATGAGGGACAGCTGGGACTGGGGGACACTGAGGAAAGGAATACCT TTCACGTGATCTCCTTCTTTACATCTGAGCATAAGATCAAGCAGCTGAG CGCCGGCTCCAACACATCTGCAGCCCTGACTGAGGACGGGCGCCTGTTC ATGTGGGGAGATAATTCAGAGGGCCAGATTGGGCTGAAAAACGTGAGC AACGTGTGCGTGCCTCAGCAGGTGACCATCGGAAAGCCAGTCAGTTGG ATTTCATGTGGCTACTATCATAGCGCCTTCGTGACCACAGATGGCGAGC TGTACGTCTTTGGGGAGCCCGAAAACGGAAAACTGGGCCTGCCTAACC AGCTGCTGGGCAATCACCGGACACCCCAGCTGGTGTCCGAGATCCCTGA AAAAGTGATCCAGGTCGCCTGCGGGGGAGAGCATACAGTGGTCCTGAC

[0297]

[0298] TGAGAATGCCGTGTACACCTTCGGACTGGGCCAGTTTGGCCAGCTGGGG CTGGGAACCTTCCTGTTTGAGACATCCGAACCAAAAGTGATCGAGAACA TTCGCGACCAGACTATCAGCTACATTTCCTGCGGAGAGAATCACACCGC ACTGATCACAGACATTGGCCTGATGTATACCTTTGGCGATGGGCGGCAC GGGAAGCTGGGACTGGGCCTGGAGAACTTCACTAATCACTTCATCCCCA CCCTGTGCTCTAACTTCCTGCGGTTCATCGTGAAACTGGTCGCTTGCGGC GGGTGTCACATGGTGGTCTTCGCTGCACCTCATAGGGGCGTGGCTAAGG AGATCGAATTTGACGAGATTAACGATACATGCCTGAGCGTGGCAACTTT CCTGCCATACAGCTCCCTGACTTCTGGCAATGTGCTGCAGAGAACCCTG AGTGCAAGGATGCGGAGAAGGGAGAGGGAACGCTCTCCTGACAGTTTC TCAATGCGACGAACCCTGCCACCTATCGAGGGGACACTGGGACTGAGT GCCTGCTTCCTGCCTAACTCAGTGTTTCCACGATGTAGCGAGCGGAATC TGCAGGAGTCTGTCCTGAGTGAGCAGGATCTGATGCAGCCAGAGGAAC CCGACTACCTGCTGGATGAGATGACCAAGGAGGCCGAAATCGACAACT CTAGTACAGTGGAGTCCCTGGGCGAGACTACCGATATCCTGAATATGAC ACACATTATGTCACTGAACAGCAATGAGAAGAGTCTGAAACTGTCACC AGTGCAGAAGCAGAAGAAACAGCAGACTATTGGCGAGCTGACTCAGGA CACCGCCCTGACAGAGAACGACGATAGCGATGAGTATGAGGAAATGTC CGAGATGAAGGAAGGCAAAGCTTGTAAGCAGCATGTGAGTCAGGGGAT CTTCATGACACAGCCAGCCACAACTATTGAGGCTTTTTCAGACGAGGAA GTGGAGATCCCCGAGGAAAAAGAGGGCGCAGAAGATTCCAAGGGGAA TGGAATTGAGGAACAGGAGGTGGAAGCCAACGAGGAAAATGTGAAAG TCCACGGAGGCAGGAAGGAGAAAACAGAAATCCTGTCTGACGATCTGA CTGACAAGGCCGAGGTGTCCGAAGGCAAGGCAAAATCTGTCGGAGAGG CAGAAGACGGACCAGAGGGACGAGGGGATGGAACCTGCGAGGAAGGC TCAAGCGGGGCTGAGCATTGGCAGGACGAGGAACGAGAGAAGGGCGA AAAGGATAAAGGCCGCGGGGAGATGGAACGACCTGGAGAGGGCGAAA AAGAGCTGGCAGAGAAGGAGGAATGGAAGAAAAGGGACGGCGAGGAA CAGGAGCAGAAAGAAAGGGAGCAGGGCCACCAGAAGGAGCGCAACCA GGAGATGGAAGAGGGCGGCGAGGAAGAGCATGGCGAGGGAGAAGAGG AAGAGGGCGATAGAGAAGAGGAAGAGGAAAAAGAAGGCGAAGGGAA GGAGGAAGGAGAGGGCGAGGAAGTGGAAGGCGAGAGGGAAAAGGAG GAAGGAGAACGGAAGAAAGAGGAAAGAGCCGGCAAAGAGGAAAAGG GCGAGGAAGAGGGCGATCAGGGCGAAGGCGAGGAGGAAGAGACCGAG GGCCGCGGGGAAGAGAAAGAGGAGGGAGGAGAGGTGGAGGGCGGAGA GGTCGAAGAGGGAAAGGGCGAGCGCGAAGAGGAAGAGGAAGAGGGCG AGGGCGAGGAAGAAGAGGGCGAGGGGGAAGAAGAGGAGGGAGAGGG CGAAGAGGAAGAGGGGGAGGGAAAGGGCGAAGAGGAAGGAGAGGAA GGGGAGGGAGAGGAAGAGGGGGAGGAGGGCGAGGGGGAAGGCGAGG AGGAAGAAGGAGAGGGGGAAGGCGAAGAGGAAGGCGAGGGGGAAGG AGAGGAGGAAGAAGGGGAAGGCGAAGGCGAAGAGGAGGGAGAAGGA GAGGGGGAGGAAGAGGAAGGAGAAGGGAAGGGCGAGGAGGAAGGCG AAGAGGGAGAGGGGGAAGGCGAGGAAGAGGAAGGCGAGGGCGAAGG AGAGGACGGCGAGGGCGAGGGAGAAGAGGAGGAAGGGGAATGGGAA GGCGAAGAAGAGGAAGGCGAAGGCGAAGGCGAAGAAGAGGGCGAAG GGGAGGGCGAGGAGGGCGAAGGCGAAGGGGAGGAAGAGGAAGGCGA AGGAGAAGGCGAGGAAGAAGAGGGAGAGGAGGAAGGCGAGGAGGAA GGAGAGGGGGAGGAGGAGGGAGAAGGCGAGGGCGAAGAAGAAGAAG AGGGAGAAGTGGAGGGCGAAGTCGAGGGGGAGGAGGGAGAAGGGGAA GGGGAGGAAGAAGAGGGCGAAGAAGAAGGCGAGGAAAGAGAAAAAG AGGGAGAAGGCGAGGAAAACCGGAGAAATAGGGAAGAGGAGGAAGA GGAAGAGGGAAAGTACCAGGAGACAGGCGAAGAGGAAAACGAGCGGC AGGATGGCGAGGAATATAAGAAAGTGAGCAAGATCAAAGGATCCGTCA AGTACGGCAAGCACAAAACCTATCAGAAGAAAAGCGTGACCAACACAC AGGGGAATGGAAAAGAGCAGCGAAGTAAAATGCCTGTGCAGTCAAAAC GGCTGCTGAAGAATGGCCCAAGCGGGTCTAAAAAATTCTGGAACAATG

[0299]

[0300] TCCTGCCACACTATCTGGAACTGAAG

[0301] 52 WT RPGR ATGAGGGAGCCGGAAGAGCTGATGCCCGATTCGGGTGCTGTGTTTACAT Transgene TTGGGAAAAGTAAATTTGCTGAAAATAATCCCGGTAAATTCTGGTTTAA AAATGATGTCCCTGTACATCTTTCATGTGGAGATGAACATTCTGCTGTTG TTACCGGAAATAATAAACTTTACATGTTTGGCAGTAACAACTGGGGTCA GTTAGGATTAGGATCAAAGTCAGCCATCAGCAAGCCAACATGTGTCAA AGCTCTAAAACCTGAAAAAGTGAAATTAGCTGCCTGTGGAAGGAACCA CACCCTGGTGTCAACAGAAGGAGGCAATGTATATGCAACTGGTGGAAA TAATGAAGGACAGTTGGGGCTTGGTGACACCGAAGAAAGAAACACTTT TCATGTAATTAGCTTTTTTACATCCGAGCATAAGATTAAGCAGCTGTCTG CTGGATCTAATACTTCAGCTGCCCTAACTGAGGATGGAAGACTTTTTAT GTGGGGTGACAATTCCGAAGGGCAAATTGGTTTAAAAAATGTAAGTAA TGTCTGTGTCCCTCAGCAAGTGACCATTGGGAAACCTGTCTCCTGGATC TCTTGTGGATATTACCATTCAGCTTTTGTAACAACAGATGGTGAGCTAT ATGTGTTTGGAGAACCTGAGAATGGGAAGTTAGGTCTTCCCAATCAGCT CCTGGGCAATCACAGAACACCCCAGCTGGTGTCTGAAATTCCGGAGAA GGTGATCCAAGTAGCCTGTGGTGGAGAGCATACTGTGGTTCTCACGGAG AATGCTGTGTATACCTTTGGGCTGGGACAATTTGGTCAGCTGGGTCTTG GCACTTTTCTTTTTGAAACTTCAGAACCCAAAGTCATTGAGAATATTAG GGATCAAACAATAAGTTATATTTCTTGTGGAGAAAATCACACAGCTTTG ATAACAGATATCGGCCTTATGTATACTTTTGGAGATGGTCGCCACGGAA AATTAGGACTTGGACTGGAGAATTTTACCAATCACTTCATTCCTACTTTG TGCTCTAATTTTTTGAGGTTTATAGTTAAATTGGTTGCTTGTGGTGGATG TCACATGGTAGTTTTTGCTGCTCCTCATCGTGGTGTGGCAAAAGAAATT GAATTCGATGAAATAAATGATACTTGCTTATCTGTGGCGACTTTTCTGCC GTATAGCAGTTTAACCTCAGGAAATGTACTGCAGAGGACTCTATCAGCA CGTATGCGGCGAAGAGAGAGGGAGAGGTCTCCAGATTCTTTTTCAATGA GGAGAACACTACCTCCAATAGAAGGGACTCTTGGCCTTTCTGCTTGTTT TCTCCCCAATTCAGTCTTTCCACGATGTTCTGAGAGAAACCTCCAAGAG AGTGTCTTATCTGAACAGGACCTCATGCAGCCAGAGGAACCAGATTATT TGCTAGATGAAATGACCAAAGAAGCAGAGATAGATAATTCTTCAACTG TAGAAAGCCTTGGAGAAACTACTGATATCTTAAACATGACACACATCAT GAGCCTGAATTCCAATGAAAAGTCATTAAAATTATCACCAGTTCAGAAA CAAAAGAAACAACAAACAATTGGGGAACTGACGCAGGATACAGCTCTT ACTGAAAACGATGATAGTGATGAATATGAAGAAATGTCAGAAATGAAA GAAGGGAAAGCATGTAAACAACATGTGTCACAAGGGATTTTCATGACG CAGCCAGCTACGACTATCGAAGCATTTTCAGATGAGGAAGTAGAGATC CCAGAGGAGAAGGAAGGAGCAGAGGATTCAAAAGGAAATGGAATAGA GGAGCAAGAGGTAGAAGCAAATGAGGAAAATGTGAAGGTGCATGGAG GAAGAAAGGAGAAAACAGAGATCCTATCAGATGACCTTACAGACAAAG CAGAGGTGAGTGAAGGCAAGGCAAAATCAGTGGGAGAAGCAGAGGAT GGGCCTGAAGGTAGAGGGGATGGAACCTGTGAGGAAGGTAGTTCAGGA GCAGAACACTGGCAAGATGAGGAGAGGGAGAAGGGGGAGAAAGACAA GGGTAGAGGAGAAATGGAGAGGCCAGGAGAGGGAGAGAAGGAACTAG CAGAGAAGGAAGAATGGAAGAAGAGGGATGGGGAAGAGCAGGAGCAA AAGGAGAGGGAGCAGGGCCATCAGAAGGAAAGAAACCAAGAGATGGA GGAGGGAGGGGAGGAGGAGCATGGAGAAGGAGAAGAAGAGGAGGGA GACAGAGAAGAGGAAGAAGAGAAGGAGGGAGAAGGGAAAGAGGAAG GAGAAGGGGAAGAAGTGGAGGGAGAACGTGAAAAGGAGGAAGGAGA GAGGAAAAAGGAGGAAAGAGCGGGGAAGGAGGAGAAAGGAGAGGAA GAAGGAGACCAAGGAGAGGGGGAAGAGGAGGAAACAGAGGGGAGAG GGGAGGAAAAAGAGGAGGGAGGGGAAGTAGAGGGAGGGGAAGTAGA GGAGGGGAAAGGAGAGAGGGAAGAGGAAGAGGAGGAGGGTGAGGGG GAAGAGGAGGAAGGGGAGGGGGAAGAGGAGGAAGGGGAGGGGGAAG AGGAGGAAGGAGAAGGGAAAGGGGAGGAAGAAGGGGAAGAAGGAGA AGGGGAGGAAGAAGGGGAGGAAGGAGAAGGGGAGGGGGAAGAGGAG GAAGGAGAAGGGGAGGGAGAAGAGGAAGGAGAAGGGGAGGGAGAAG AGGAGGAAGGAGAAGGGGAGGGAGAAGAGGAAGGAGAAGGGGAGGG

[0302]

[0303] AGAAGAGGAGGAAGGAGAAGGGAAAGGGGAGGAGGAAGGAGAGGAA GGAGAAGGGGAGGGGGAAGAGGAGGAAGGAGAAGGGGAAGGGGAGG ATGGAGAAGGGGAGGGGGAAGAGGAGGAAGGAGAATGGGAGGGGGA AGAGGAGGAAGGAGAAGGGGAGGGGGAAGAGGAAGGAGAAGGGGAA GGGGAGGAAGGAGAAGGGGAGGGGGAAGAGGAGGAAGGAGAAGGGG AGGGGGAAGAGGAGGAAGGGGAAGAAGAAGGGGAGGAAGAAGGAGA GGGAGAGGAAGAAGGGGAGGGAGAAGGGGAGGAAGAAGAGGAAGGG GAAGTGGAAGGGGAGGTGGAAGGGGAGGAAGGAGAGGGGGAAGGAG AGGAAGAGGAAGGAGAGGAGGAAGGAGAAGAAAGGGAAAAGGAGGG GGAAGGAGAAGAAAACAGGAGGAACAGAGAAGAGGAGGAGGAAGAA GAGGGGAAGTATCAGGAGACAGGCGAAGAAGAGAATGAAAGGCAGGA TGGAGAGGAGTACAAAAAAGTGAGCAAAATAAAAGGATCTGTGAAATA TGGCAAACATAAAACATATCAAAAAAAGTCAGTTACTAACACACAGGG AAATGGGAAAGAGCAGAGGTCCAAAATGCCAGTCCAGTCAAAACGACT TTTAAAAAACGGGCCATCAGGTTCCAAAAAGTTCTGGAATAATGTATTA

[0304]

[0305] CCACATTACTTGGAATTGAAG

[0306] 54 EXAMPLES

[0307] Example 1 - Clinical trial study design

[0308] A clinical study was undertaken to evaluate the safety and efficacy of a recombinant adeno-associated virus vector (GRK1-RPGR vector packaged in an AAV2-derived capsid) in patients with X-linked retinitis pigmentosa caused by RPGR mutations. The vector comprised a vector genome as shown in the schematic in Fig. 1 and having the sequence of SEQ ID NO: 8 (containing the RPGR transgene sequence of SEQ ID NO: 2 / 18), and the AAV2-derived capsid was a capsid comprising VP1 polypeptides of sequence SEQ ID NO: 9.

[0309] The purpose of the study was to evaluate the efficacy, safety, and tolerability of two dose levels of the GRK1-RPGR vector packaged in an AAV2-derived capsid of SEQ ID NO: 9 in male subjects between 8 - 50 years of age (inclusive) with XLRP genetically confirmed by at least one pathogenic variant in the RPGR gene. 14 patients were enrolled and randomised into one of two treatment groups: Group 1 (low dose 7.5 x 1010vg / eye; N=6) and Group 2 (high dose 6.8 x 1011vg / eye; N=8). After baseline screening, each patient received the assigned dose of vector in one eye (“study eye”) via sub-retinal injection to the central macula, on a single occasion. The other eye was left untreated as a control (“fellow eye”). To prevent postoperative ocular inflammation the patients received oral corticosteroid (prednisone or prednisolone) three days prior to surgery, continuing for five days before commencing a tapering-off over the subsequent 4-6 weeks.

[0310] Patient inclusion and exclusion criteria

[0311] Inclusion Criteria:

[0312] • Provide written informed consent or assent (per local regulation), prior to the conduct of any study -related procedure. Subjects who provide assent must have a parent, guardian, or legal representative provide written informed consent.

[0313] 55 • Be between 8 and 50 years of age (inclusive) at the time of informed consent and assent (as applicable).

[0314] • Be male and have at least one documented pathogenic or likely pathogenic variant in the RPGR gene

[0315] • Have a clinical diagnosis of XLRP.

[0316] • Have a BCVA no better than 75 letters and no worse than 35 letters based on an ETDRS chart at each screening visit.

[0317] • Be able to perform all tests of visual and retinal function and structure in both eyes based on the subject's reliability, and fixation, per the investigator's discretion.

[0318] • Have detectable baseline mean macular sensitivity measured by (MAIA) microperimetry, as determined by the investigator and confirmed by the Central Reading Center (CRC).

[0319] • Have detectable EZ line in both eyes as assessed by SD-OCT and confirmed by the CRC.

[0320] Exclusion Criteria:

[0321] • Have other known disease-causing mutations documented in the subject's medical history or identified through a retinal dystrophy gene panel, that in the opinion of the investigator would interfere with the potential therapeutic effect of the study agent or the quality of the assessments.

[0322] • For subjects with herpes simplex virus (HSV):

[0323] • Have history of oral or genital herpes and unable and / or unwilling to utilize prophylactic antiviral medication.

[0324] • Have a history of ocular herpes.

[0325] • Have active oral or genital herpes or are currently receiving treatment for HSV infection.

[0326] • Have complicating systemic diseases (e.g., medical conditions causing immunosuppression, active systemic infection) that would preclude the gene transfer or ocular surgery.

[0327] • Have known sensitivity or allergy to systemic corticosteroids or other immunosuppressive medications.

[0328] • Have used anti -coagulant agents that may alter coagulation

[0329] 56 • Have received any vaccination / immunization within 28 days prior to screening and / or during screening with the exception of the influenza vaccine, which is only exclusionary if they have received the influenza vaccine within 28 days prior to randomization.

[0330] • Have used systemic corticosteroids or other immunosuppressive medications within 3 months prior to screening and / or intend to use during screening.

[0331] • Have previously received any AAV gene therapy product, stem cell therapy, cell-based therapy, or similar biologies.

[0332] Ocular Exclusion Criteria (Either Eye):

[0333] • Have pre-existing eye conditions that would preclude the planned surgery, interfere with the interpretation of study endpoints, or increase the risk of surgical complications • Have significant media opacity impacting evaluation of the retina or vitreous.

[0334] • Had intraocular surgery within 90 days of study treatment administration.

[0335] • Have any active ocular / intraocular infection or inflammation

[0336] • Have a history of steroid-induced raised IOP of >25 mmHg following corticosteroid exposure, despite topical lOP-lowering pharmacologic therapy.

[0337] • Have any artificial retinal implant or prosthesis.

[0338] • Have absence of clear ocular media and / or inadequate pupil dilation to facilitate good quality OCT images.

[0339] • Have any history of rhegmatogenous retinal detachment.

[0340] • Have myopia (spherical equivalent) exceeding -10 diopters (or axial length of >30 mm if PI deems it appropriate to measure) or presence of pathologic myopia in the study eye.

[0341] • Have passed the Low Contrast Ora-VNC™ mobility course in either eye or binocularly at any screening visit.

[0342] Patients were assessed at follow-ups every 3 months during the first year and every six months in the second year after dosing in order to monitor progress of the study and fellow eyes. The efficacy endpoints included: • The difference in the proportion of responding eyes between treated and control eyes in the low dose group and high dose group at Month 12, as measured by macular integrity assessment (MAIA) microperimetry, where response is defined as a 7dB or more improvement in at least 5 loci;

[0343] 5 • CFB (change from baseline) at Month 12 in mean sensitivity by microperimetry (MAIA);

[0344] • CFB (change from baseline) at Month 12 in mobility score assessed by the Ora-VNC™ mobility course, wherein response is defined as improvement of greater than or equal to 2 luminance levels.

[0345] 10

[0346] Example 2 - Safety (Example 1 trial)

[0347] Tables 1 and 2 show ocular serious adverse events (SAEs) and ocular treatment-emergent 15 adverse events (TEAEs) at Month 24. No ocular SAEs were deemed related to the GRK1- RPGR vector packaged in an AAV2-derived capsid of SEQ ID NO: 9. Ocular TEAEs were mostly non-serious, mild or moderate in severity, and rates were similar between high dose and low dose groups. Ocular TEAEs related to the GRK1-RPGR vector packaged in an AAV2-derived capsid of SEQ ID NO: 9 were considered mild or moderate in severity and 20 most ocular TEAEs related to the injection procedure were considered mild or moderate in severity.

[0348] Ocular Serious Low dose (n=6) N High dose (n=8) N All patients (n=14) N Adverse Events

[0349] (SAE)

[0350] Study eye Fellow eye Study eye Fellow eye Study eye Fellow eye # of patients with 2 0 0 0 2 0 any ocular SAE

[0351] Glaucoma* 1 0 0 0 1 0 Visual 1 0 0 0 1 0

[0352] Impairment**

[0353]

[0354] *Related to protocol required corticosteroids; severe; treated with medication; resolved by Study Day 181

[0355] **Related to injection procedure; ongoing

[0356]

[0357] Table 1: Study SAEs

[0358] Ocular Treatment- Low dose (n=6) N High dose (n=8) N All patients (n=14) N emergent Adverse

[0359] Events (TEAE)

[0360] Study eye Fellow eye Study eye Fellow eye Study eye Fellow eye # of patients with 3 0 2 0 5 0 any ocular TEAE

[0361] related to study

[0362] vector

[0363] Vitritis 1 0 2 0 3 0 Eye pain 1 0 0 0 1 0 Metamorphopsia 1 0 0 0 1

[0364] Photop si a 1 0 0 0 1

[0365] Visual acuity 1 0 0 0 1

[0366] reduced

[0367]

[0368] 5 Table 2: Study TEAEs

[0369] Example 3 - Efficacy (Example 1 trial)

[0370] 10

[0371] Retinal sensitivity (MAIA microperimetry)

[0372] Microperimetry as a methodology and endpoint is reviewed in Yang et al (Yesa Yang, Hannah Dunbar; Clinical Perspectives and Trends: Microperimetry as a Trial Endpoint in 15 Retinal Disease. Ophthalmologica 2 November 2021; 244 (5): 418-450). MAIA microperimetry testing of patients under mesopic conditions was performed to assess photoreceptor function for both the study eye and fellow eye (at baseline and) post-treatment at Months 1, 3, 6, 9, 12, 18 and 24. Starting at Month 3, the testing was performed by an examiner who did not know which eye received treatment (i.e., the study eye).

[0373] The primary efficacy endpoint was the proportion of responding eyes at Month 12, where a responder was defined as a 7dB or more improvement in retinal sensitivity for at least 5 loci, while a key secondary efficacy outcome measure was the change from baseline (CFB) in mean retinal sensitivity.

[0374] Fig.2 shows that at Month 12 a clear difference in response between the two dose groups was evident. While no low-dosed eyes were responsive at Month 12, 5 / 8 (63%) high dose-treated eyes showed response (only one of the corresponding fellow (untreated) eyes across both groups, was a responder). A consistent effect was observed at Month 24, although subject to skewing from loss-to-follow-up of one high-dose and two low-dose patients (Figure 2A). 25% and 57% of low- and high-dosed eyes were responders at Month 24, with no fellow eyes showing response (Figure 2B).

[0375] The positive efficacy results for a 7 dB or more improvement in at least 5 loci in the whole grid are aligned and consistent with mean sensitivity outcomes, shown in Fig.3. Mean changes from baseline over time in mean retinal sensitivity for loci within the whole grid showed a clear dose response in favour of the high dose group.

[0376] Fig.4 is a composite example of a responder participant in the high dose group as demonstrated by MAIA microperimetry heat maps and retinal sensitivity change by plotting the individual loci within the whole grid. Figure 4A shows the heat maps for the treated and untreated eyes. Positive visual sensitivity change is evident in the treated eye, increasing in both magnitude of effect as well as the overall area of effect from baseline to Month 12. In Figure 4B, there are at least 5 loci at Month 12 and, again, at Month 24 with a 7 dB or greater improvement in the study. A marked difference is also seen between the treated and untreated eye in this participant. Functional vision assessment by Ora-Visual Navigation Challenge (VNC™)

[0377] The VNC™ is a 360-degree visual challenge environment through which a patient must navigate, providing an assessment of functional vision. The VNC™ is described in Aron Shapiro, Peter Corcoran, Christian Sundstrom, Endri Angjeli, John David Rodriguez, Mark B Abelson, David A Hollander; Development and Validation of a Portable Visual Navigation Challenge for Assessment of Retinal Disease in Multi -Centered Clinical Trials. Invest.

[0378] Ophthalmol. Vis. Sci. 2017;58(8):3290.

[0379] Despite an incomplete data set, the VNC™ assessment demonstrated positive trends in the high dose group, with 5 / 8 treated eyes available for assessment showing at least a one level improvement in the VNC™, relative to 0 / 8 of the available untreated fellow eyes at month 12 (Fig. 5A), and 5 / 7 treated eyes available for assessment showing at least a one level improvement in the VNC™, relative to 1 / 6 of the available untreated fellow eyes at month 24 (Fig.5B).

[0380] Example 4 - Clinical trial study design

[0381] A further clinical study is being undertaken to evaluate the safety and efficacy of a recombinant adeno-associated virus vector (GRK1-RPGR vector packaged in an AAV2-derived capsid of SEQ ID NO: 9) in patients with X-linked retinitis pigmentosa caused by RPGR mutations. As in the Example 1 study, the vector comprises a vector genome as shown in the schematic in Fig. 1 and having the sequence of SEQ ID NO: 8 (containing the RPGR transgene sequence of SEQ ID NO: 2 / 18), encapsidated in an AAV capsid comprising VP1 polypeptides of sequence SEQ ID NO: 9. The vector composition for this trial was produced by a downstream manufacturing process comprising an anion exchange (AEX) chromatography step, resulting in a product possessing improved quality characteristics including an increased full:empty capsid ratio (at least 70%), i.e. containing a reduced number of empty capsids present relative to the number of full capsids.

[0382] The vector composition is provided below:

[0383] (a) equal to or greater than 2.5 x 1011vg / ml of the viral particles;

[0384] 61 (b) between 6 and 7 mg / ml sodium chloride;

[0385] (c) between 0.5 and 1 mg / ml potassium chloride;

[0386] (d) between 0.4 and 0.6 mg / ml calcium chloride dihydrate;

[0387] (e) between 0.2 and 0.4 mg / ml magnesium chloride hexahydrate;

[0388] (f) between 3.5 and 4.5 mg / ml sodium acetate trihydrate;

[0389] (g) between 1.5 and 2 mg / ml sodium citrate dihydrate; and

[0390] (h) between 0.01 and 0.02% polysorbate 20 (v / v).

[0391] The composition was stored in vials comprising a bromo-butyl stopper or a chloro-butyl stopper.

[0392] The purpose of the non-randomised, open-label study is to evaluate the safety and efficacy of two dose levels of GRK1-RPGR vector packaged in an AAV2-derived capsid of SEQ ID NO: 9 in male subjects with XLRP who have previously been treated with an AAV vector-based gene therapy which carries a full-length RPGR (ORF 15) transgene. After baseline screening, patients in Group 1 and Group 2 respectively receive the high (6.8 x 1011vg / eye) and low (3.7 x 1011vg / eye) dose, via a single sub-retinal injection in the eye (“study eye”) that was not previously treated (the previously-treated eye, which is not treated in this study, being referred to for the purposes of this Example as the “fellow eye”). To prevent post-operative ocular inflammation the patients receive oral corticosteroid three days prior to surgery, continuing for five days before commencing a tapering-off over the subsequent 4-6 weeks.

[0393] Patient inclusion and exclusion criteria

[0394] Inclusion Criteria:

[0395] • Be ≥12 years of age.

[0396] • Have one eye previously treated with an AAV vector-based gene therapy designed to provide full-length functioning RPGR protein.

[0397] • Have a BCVA no better than 78 letters and no worse than 34 letters.

[0398] • Be able to perform all tests of visual and retinal function and structure in both eyes based on the participant's reliability and fixation, per the Investigator's discretion. • Have detectable baseline mean macular sensitivity measured by MAIA microperimetry, as determined by the Investigator and confirmed by the Central Reading Center (CRC).

[0399] • Have detectable EZ line in the study eye as assessed by SD-OCT and confirmed by the CRC.

[0400] Exclusion Criteria:

[0401] • Have other known disease-causing mutations documented in the participant's medical history or identified through a retinal dystrophy gene panel that, in the opinion of the Investigator, would interfere with the potential therapeutic effect of the study agent or the quality of the assessments.

[0402] • Have pre-existing eye conditions that would preclude the planned surgery, interfere with the interpretation of study endpoints, or increase the risk of surgical complications • Had intraocular surgery within 90 days of study treatment administration.

[0403] • Have any active ocular / intraocular infection or inflammation

[0404] • Have a history of steroid-induced raised IOP of >25 mmHg following corticosteroid exposure, despite topical lOP-lowering pharmacologic therapy.

[0405] Patients are assessed at Months 1 (30 days), 3, 6, 9 and 12 (and then less frequent longer-term follow-up) following administration of the study drug in order to monitor progress of the study and fellow eyes. The efficacy endpoints include:

[0406] • Proportion of responding eyes in treated versus control eyes at Month 12 where responder is defined as an improvement of at least 15-letters on low-luminance visual acuity (LEVA);

[0407] • CFB (change from baseline) at Month 12 in Low Luminance Visual Acuity (LLVA) using Early-Treatment Diabetic Retinopathy Study (ETDRS) visual acuity;

[0408] • CFB (change from baseline) at Month 12 in Best Corrected Visual Acuity (BCVA) using Early-Treatment Diabetic Retinopathy Study (ETDRS) visual acuity;

[0409] • CFB (change from baseline) at Month 12 in mean sensitivity across the whole grid by microperimetry (MAIA); • Response, as measured by MAIA microperimetry, where response is defined as a greater than or equal to 7 decibel (dB) visual sensitivity improvement from baseline in at least 5 loci at Month 12;

[0410] • CFB (change from baseline) at Month 12 in mobility score assessed by the Ora-VNC™ mobility course.

[0411] Example 5 - Safety (Example 4 trial)

[0412] Table 3 shows that ocular treatment-emergent adverse events (TEAEs) at Month 3 were mostly non-serious, mild or moderate in severity. No TEAEs were deemed related to the study drug, and no ocular inflammatory AEs were reported.

[0413] Ocular Treatment- High dose (n=7) N Low dose (n=3) All patients (n=10) emergent Adverse Events N N

[0414] (TEAE)

[0415] Study Fellow Study Fellow Study eye Fellow eye eye* eye eye* eye* Serious Glaucoma** 0 0 1 0 1 0 Moderate Eye pain, 2 0 2 0 4 0

[0416] injection

[0417] related

[0418] Eye pain, 1 0 0 0 1 0 corticosteroid- related

[0419] IOP increased 1 0 2 1 3 1 Vision blurred 1 0 0 0 1 0

[0420]

[0421] *Fellow eye = eyes previously treated with an AAV gene therapy carrying a full-length RPGR ORF15 gene

[0422] ** Severe and serious; related to protocol-required corticosteroids

[0423] All patients received standard dose corticosteroid regimen Table 3: Ocular TEAEs

[0424] Example 6 - Efficacy (Example 4 trial)

[0425] Functional vision assessment by low luminance visual acuity (LLVA)

[0426] The use of LLVA as a methodology for assessing visual function in ophthalmological clinical trials is reviewed in Wood LJ, Jolly JK, Buckley TMW, Josan AS, & MacLaren RE; Low luminance visual acuity as a clinical measure and clinical trial outcome measure: a scoping review. Ophthalmic Physiol Opt. 2021; 41: 213-223. In this study, LLVA testing of patients is performed by using a neutral density filter to reduce the luminance by 2 log units relative to that used for BCVA measurement.

[0427] Fig. 6 shows mean change from baseline LLVA data through to Month 3 for Group 1 and 2 study eyes, and the fellow eyes (combined Groups 1 and 2). While the fellow (i.e. previously-treated) eyes remained stable over the period, significant increases were observed for both groups of study eyes.

[0428] Fig. 7 presents Month 3 results for the proportion of study and fellow eyes falling into LLVA response categories (<10 ETDRS letters; 10-14 letters; > 15 letters), respectively plotted against baseline low luminance deficit (LLD) (Fig.7A), baseline BCVA (Fig.7B) and baseline LLVA (Fig.7C). The study eye data in each case are ordered as waterfall, with the fellow eye data ordered on a corresponding patient basis as indicated by the notation at the top of each bar. LLD is the difference between the BCVA and LLVA values. An apparent correlation between high baseline LLD and high LLVA response is evident from Fig.7A. It is also notable that Figs. 7B and 7C show that average BCVA and LLVA scores for the fellow eyes are at least comparable to the average scores for the study eyes. At the time of previous treatment of the fellow eyes, typically a number of years prior to the present treatment of the study eyes, the fellow eyes were the lower-scoring of each patient’s eyes. At the time of this study, the previously-treated fellow eyes are not, on average, scoring lower than the study eyes at 3 months following treatment.

[0429] 65 Fig. 8 shows mean change from baseline LLVA data through to Month 9 for Group 1 and 2 study eyes, and the fellow eyes (combined Groups 1 and 2). While the fellow (i.e. previously-treated) eyes showed only minor improvement over the period, significant increases were observed for both groups of study eyes, in particular reaching an average increase of 16 letters by month 9.

[0430] Fig. 9 presents Month 6 to Month 12 (based on the latest study visit for each patient) results for the proportion of study and fellow eyes falling into LLVA response categories (<10 ETDRS letters; 10-14 letters; > 15 letters), respectively plotted against baseline low luminance deficit (LLD). The study eye data in each case are ordered as waterfall, with the fellow eye data ordered on a corresponding patient basis as indicated by the notation at the top of each bar. LLD is the difference between the BCVA and LLVA values. An apparent correlation between high baseline LLD and high LLVA response is evident from Fig. 9. Fig. 10 presents Month 6 to Month 12 (based on the latest study visit for each patient) individual LLVA results for each study and corresponding fellow eye. The data shows that 57% of the study eyes in the high dose group show a >15 letter increase in LLVA score, while 86% show an increase of at least 10 letters. 100% of the low dose group showed at increase of at least 10 letters.

[0431] Fig. 11 shows exemplary MAIA microperimetry data for a patient who took part in the Example 1 trial (Eye 1) and the Example 4 trial (Eye 2). As shown in Figs. 11B and 11C, both eyes showed a greater than or equal to 7 dB improvement from baseline at at least 5 loci at Month 12 relative to baseline.

[0432] Example 7 - Clinical trial study design

[0433] A further clinical study is being undertaken to evaluate the safety and efficacy of a recombinant adeno-associated virus vector (GRK1-RPGR vector packaged in an AAV2-derived capsid of SEQ ID NO: 9) in patients with X-linked retinitis pigmentosa caused by RPGR mutations. As in the Examples 1 and 4 studies, the vector comprises a vector genome as shown in the schematic in Fig. 1 and having the sequence of SEQ ID NO: 8 (containing the RPGR transgene sequence of SEQ ID NO: 2 / 18), encapsidated in an AAV capsid comprising VP1 polypeptides of sequence SEQ ID NO: 9. As in the Example 4 study, the vector composition for this trial was produced by a downstream manufacturing process comprising an anion exchange (AEX) chromatography step, resulting in a product possessing improved quality characteristics including an increased full:empty capsid ratio (at least 70%), i.e. containing a reduced number of empty capsids present relative to the number of full capsids.

[0434] The vector composition is provided below:

[0435] (a) equal to or greater than 2.5 x 1011vg / ml of the viral particles;

[0436] (b) between 6 and 7 mg / ml sodium chloride;

[0437] (c) between 0.5 and 1 mg / ml potassium chloride;

[0438] (d) between 0.4 and 0.6 mg / ml calcium chloride dihydrate;

[0439] (e) between 0.2 and 0.4 mg / ml magnesium chloride hexahydrate;

[0440] (f) between 3.5 and 4.5 mg / ml sodium acetate trihydrate;

[0441] (g) between 1.5 and 2 mg / ml sodium citrate dihydrate; and

[0442] (h) between 0.01 and 0.02% polysorbate 20 (v / v).

[0443] The purpose of the randomised, controlled, dose-masked, multi-centre study is to evaluate and compare two dose levels of GRK1-RPGR vector packaged in an AAV2-derived capsid of SEQ ID NO: 9 with an untreated control group, in male subjects with XLRP. Approximately 75 eligible male participants between 12 and 50 years of age (inclusive) will be randomized in a 1: 1: 1 ratio to 1 of 3 groups. The randomization will be stratified by age (<18 years vs >18 years). After baseline screening, patients in Group 1 and Group 2 respectively will receive the high (6.8 x 1011vg / eye) and low (3.7 x 1011vg / eye) dose, via a single sub-retinal injection. Patients in Group 3 will not be treated, but will otherwise be screened and evaluated in the same way as Groups 1 and 2. One eye will be selected as the study eye for each participant based on pre-determined criteria. To prevent post-operative ocular inflammation the patients will receive prophylactic pre-, peri- and post-surgical corticosteroid medications including oral, topical and sub-tenon corticosteroid.

[0444] Patient inclusion and exclusion criteria Inclusion Criteria:

[0445] • Be between 12 and 50 years of age.

[0446] • Be male (XY chromosome) and have at least one documented pathogenic or likely pathogenic variant in the RPGR gene within exons 1-14 and / or ORF15 from an appropriately certified or accredited laboratory.

[0447] • Have a clinical diagnosis of XLRP.

[0448] • Have a BCVA of <78 letters and >34 letters in the study eye.

[0449] • Have a LLVA <64 letters in the study eye.

[0450] • Have an LLD of >10 in the study eye.

[0451] • Have detectable baseline mean macular sensitivity measured by MAIA microperimetry, between 1-12 decibels (dB) in the study eye.

[0452] • Have a detectable sub-foveal EZ line in the study eye.

[0453] Exclusion Criteria:

[0454] • Have other known disease-causing mutations documented in the participant's medical history or identified through a retinal dystrophy gene panel that, in the opinion of the Investigator, would interfere with the potential therapeutic effect of the study agent or the quality of the assessments.

[0455] • Have pre-existing eye conditions that would preclude the planned surgery, interfere with the interpretation of study endpoints, or increase the risk of surgical complications. • Had intraocular surgery within 90 days of study treatment administration.

[0456] • Have any active ocular / intraocular infection or inflammation.

[0457] • Have a history of steroid-induced raised IOP of >25 mmHg following corticosteroid exposure, despite topical lOP-lowering pharmacologic therapy.

[0458] • Have an artificial retinal implant or prosthesis.

[0459] • Have absence of clear ocular media and / or inadequate pupil dilation to facilitate good quality SD-OCT images.

[0460] • Have any history of rhegmatogenous retinal detachment.

[0461] • Have myopia (spherical equivalent) exceeding -10 diopters (or axial length of >30mm if PI deems it appropriate to measure) or presence of pathological myopia in the study eye. • Have passed the Low Contrast Ora-VNC™ mobility course ≤0.35 lux light level n either eye or binocularly at any screening visit.

[0462] Patients will be assessed at Months 1 (30 days), 3, 6, 9 and 12 (and then less frequent longer-term follow-up) following (in the case of Groups 1 and 2) administration of the study drug in order to monitor progress of the study eyes. Primary and secondary efficacy endpoints will include:

[0463] • Proportion of participants with a ≥10 letters increase from baseline in LLVA at Month 12;

[0464] • Proportion of participants with a ≥15 letter increase from baseline in LLVA at Month 12;

[0465] • Change from baseline in LLVA at Month 12;

[0466] • Change from baseline in mean sensitivity across the whole grid, as measured by MAIA microperimetry, at Month 12;

[0467] • Change from baseline in full-field stimulus threshold (FST) at Month 12;

[0468] • Change from baseline in mobility test score at Month 12, 18, and 24 as measured by the Ora-VNC™ mobility course;

[0469] • Change from baseline in mean sensitivity across the whole grid, as measured by MAIA microperimetry at Month 18 and 24;

[0470] • Response at Month 12, 18, and 24 as measured by MAIA microperimetry, where response is defined as a >7 decibel (dB) visual sensitivity improvement from baseline in at least 5 loci;

[0471] • Change from baseline in mobility test score at Month 12, 18, and 24 as measured by the Mobility Standardized Test- Virtual Reality (MOST-VR) mobility course;

[0472] • Change from baseline in mean sensitivity across the central 4, central 16, and central 36 loci, respectively, as measured by MAIA microperimetry, at Month 12, 18 and 24;

[0473] • Proportion of participants with a ≥15 letter increase from baseline in LLVA at Month 18 and 24;

[0474] • Change in low luminance deficit (LLD), defined as the difference between BCVA and LLVA at Month 12, 18, and 24; • Change from baseline in BCVA over time as assessed by an Early Treatment of Diabetic Retinopathy Study (ETDRS) or Tumbling E chart at Month 12, 18, and 24;

[0475] • Change from baseline in FST at Month 24;

[0476] Proportion of participants with a ≥10 letter increase from baseline in LLVA at Month 18 and 24.

[0477] 70 ASPECTS

[0478] 1. A polynucleotide comprising two inverted terminal repeats (ITRs), an hGRKl promoter, an SV40 late intron, a transgene encoding an RPGR polypeptide, and an SV40 late polyA.

[0479] 2. The polynucleotide of aspect 1, wherein the hGRKl promoter comprises a sequence that is:

[0480] (a) between 200 and 300 nucleotides in length;

[0481] (b) between 250 and 300 nucleotides in length; or

[0482] (c) 295 nucleotides in length.

[0483] 3. The polynucleotide of aspect 1 or aspect 2, wherein the hGRKl promoter comprises a sequence that is identical to SEQ ID NO: 10, but for:

[0484] (a) a T to G substitution at position 199;

[0485] (b) a C to A substitution at position 245; and / or

[0486] (c) a GGC insertion between positions 246 and 247.

[0487] 4. The polynucleotide of any one of the preceding aspects, wherein the hGRKl promoter comprises a sequence at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 1 or SEQ ID NO: 17, preferably wherein the hGRKl promoter comprises a sequence at least 90% identical to SEQ ID NO: 1.

[0488] 5. The polynucleotide of any one of the preceding aspects, wherein the hGRKl promoter comprises a sequence at least 98% identical to SEQ ID NO: 1.

[0489] 6. The polynucleotide of any one of the preceding aspects, wherein the hGRKl promoter comprises a sequence at least 99% identical to SEQ ID NO: 1.

[0490] 7. The polynucleotide of any one of the preceding aspects, wherein the hGRKl promoter comprises a sequence that is identical to SEQ ID NO: 1.

[0491] 8. The polynucleotide of any one of the preceding aspects, wherein the hGRKl promoter is operably linked to the transgene.

[0492] 71 9. The polynucleotide of any one of the preceding aspects, wherein the transgene and / or the polynucleotide is codon-optimised.

[0493] 10. The polynucleotide of aspect 9, wherein the transgene and / or the polynucleotide is codon optimised if the product of the RPGR transgene is expressed at a higher level in HEK293T cells compared to an equivalent polynucleotide comprising a corresponding RPGR transgene.

[0494] 11. The polynucleotide of any one of the preceding aspects, wherein the transgene and / or the polynucleotide comprises:

[0495] (a) more CpGs than a wild-type RPGR transgene;

[0496] (b) more than 161 CpGs; and / or

[0497] (c) about 167 CpGs.

[0498] 12. The polynucleotide of any one of the preceding aspects, wherein the transgene comprises a nucleotide sequence at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 18, preferably wherein the transgene comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 2.

[0499] 13. The polynucleotide of any one of the preceding aspects, wherein the transgene comprises a nucleotide sequence at least 98% identical to SEQ ID NO: 2.

[0500] 14. The polynucleotide of any one of the preceding aspects, wherein the transgene comprises a nucleotide sequence at least 99.5% identical to SEQ ID NO: 2.

[0501] 15. The polynucleotide of any one of the preceding aspects, wherein the transgene comprises a nucleotide sequence identical to SEQ ID NO: 2.

[0502] 16. The polynucleotide of any one of the preceding aspects, wherein the transgene corresponds to ORF 15 of RPGR (RPGR ORF 15). 17. The polynucleotide of any one of the preceding aspects, wherein the transgene comprises RPGR ORF15.

[0503] 18. The polynucleotide of any one of the preceding aspects, wherein the RPGR polypeptide is at least 1100 amino acids, optionally wherein the RPGR polypeptide comprises 1152 amino acids.

[0504] 19. The polynucleotide of any one of the preceding aspects, wherein the transgene encodes the full-length RPGR ORF 15 polypeptide.

[0505] 20. The polynucleotide of any one of the preceding aspects, wherein the transgene comprises a nucleotide sequence that is identical to SEQ ID NO: 7 but for one or more substitutions selected from a substitution to T at position 30; C at position 33; C at position 49; G at position 57; C at position 60; A at position 69; G at position 71; T at position 170; T at position 189; C at position 441; C at position 537; G at position 546; C at position 786; C at position 792; G at position 966; G at position 969; C at position 990; C at position 1011; C at position 1014; C at position 1029; G at position 1299; G at position 1689; C at position 3355; A at position 3357; A at position 3363; A at position 3403; G at position 3404; C at position 3405; G at position 3408; T at position 3409; C at position 3410; C at position 3432; A at position 3438; and G at position 3456.

[0506] 21. The polynucleotide of any one of the preceding aspects, wherein the ITRs are AAV2 ITRs.

[0507] 22. The polynucleotide of any one of the preceding aspects, wherein the ITRs comprise a 5’ ITR and a 3 ’ ITR.

[0508] 23. The polynucleotide of aspect 22, wherein the 5’ ITR comprises a sequence:

[0509] (a) at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 3; or

[0510] (b) that is identical to SEQ ID NO: 3.

[0511] 24. The polynucleotide of aspect 22 or aspect 23, wherein the 3’ ITR comprises a sequence: (a) at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 4; or

[0512] (b) that is identical to SEQ ID NO: 4.

[0513] 25. The polynucleotide of any one of the preceding aspects, wherein the SV40 late intron comprises a sequence at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 5 or SEQ ID NO: 12, preferably at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 5.

[0514] 26. The polynucleotide of any one of the preceding aspects, wherein the SV40 late intron comprises a sequence identical to SEQ ID NO: 5 or SEQ ID NO: 12, preferably SEQ ID NO: 5.

[0515] 27. The polynucleotide of any one of the preceding aspects, wherein the SV40 late intron is operably linked to the transgene.

[0516] 28. The polynucleotide of any one of the preceding aspects, wherein the SV40 late polyA comprises a sequence at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 6 or SEQ ID NO: 13, preferably at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 6.

[0517] 29. The polynucleotide of any one of the preceding aspects, wherein the SV40 late polyA comprises a sequence identical to SEQ ID NO: 6 or SEQ ID NO: 13, preferably SEQ ID NO: 6.

[0518] 30. The polynucleotide of any one of aspects 22 to 29, wherein the polynucleotide comprises the following elements in 5’ to 3’ order: the 5’ ITR, the hGRKl promoter, the SV40 late intron, the transgene encoding the RPGR polypeptide, the SV40 late polyA, and the 3’ ITR.

[0519] 31. The polynucleotide of any one of the preceding aspects, wherein the polynucleotide is:

[0520] (a) between 4000 and 5000 nucleotides in length;

[0521] (b) between 4000 and 4500 nucleotides in length;

[0522] (c) between 4250 and 4500 nucleotides in length; (d) less than 4500 nucleotides in length; or

[0523] (e) around 4480 nucleotides in length.

[0524] 32. The polynucleotide of any one of the preceding aspects, wherein the polynucleotide comprises a nucleotide sequence:

[0525] (a) at least 90%, at least 95%, or at least 98% identical to a fragment of at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, or at least 4250 nucleotides of SEQ ID NO: 8;

[0526] (b) at least 95% identical to a fragment of at least 4250 nucleotides of SEQ ID NO: 8; (c) at least 98% identical to a fragment of at least 4250 nucleotides of SEQ ID NO: 8; (d) at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 8;

[0527] (e) at least 98% identical to SEQ ID NO: 8; and / or

[0528] (f) identical to SEQ ID NO: 8.

[0529] 33. The polynucleotide of any one of the preceding aspects, wherein the polynucleotide comprises a nucleotide sequence at least 98% identical to a fragment of at least 4250 nucleotides of SEQ ID NO: 8.

[0530] 34. The polynucleotide of any one of the preceding aspects, wherein the polynucleotide comprises a nucleotide sequence at least 98% identical to SEQ ID NO: 8.

[0531] 35. The polynucleotide of any one of the preceding aspects, wherein the polynucleotide comprises a nucleotide sequence identical to SEQ ID NO: 8.

[0532] 36. A viral particle comprising a recombinant genome comprising the polynucleotide of any of the preceding aspects and a capsid.

[0533] 37. The viral particle of aspect 36, which is an AAV particle and / or the viral particle was produced in mammalian cells.

[0534] 38. The viral particle of aspect 36 or aspect 37, wherein the capsid is: (i) an AAV2-derived capsid;

[0535] (ii) not an AAV5 capsid; or

[0536] (iii) an AAV5 capsid,

[0537] preferably wherein the capsid is an AAV2-derived capsid.

[0538] 39. The viral particle of any one of aspects 36 to 38, wherein the capsid is an AAV2 capsid comprising a substitution of at least one Y residue with an F residue, optionally wherein the AAV2 capsid comprises a Y444F, Y500F, and / or Y730F substitution.

[0539] 40. The viral particle of any one of aspects 36 to 39, wherein the capsid comprises an amino acid sequence:

[0540] (a) at least 90%, at least 95%, or at least 98% identical to a fragment of at least 600, at least 650, at least 675, or at least 700 amino acids of SEQ ID NO: 9;

[0541] (b) at least 95% identical to a fragment of at least 650 amino acids of SEQ ID NO: 9; (c) at least 98% identical to a fragment of at least 700 amino acids of SEQ ID NO: 9; (d) at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 9;

[0542] (e) at least 98% identical to SEQ ID NO: 9; and / or

[0543] (f) identical to SEQ ID NO: 9.

[0544] 41. The viral particle of any one of aspects 36 to 40, wherein the capsid comprises an amino acid sequence at least 98% identical to a fragment of at least 700 amino acids of SEQ ID NO: 9.

[0545] 42. The viral particle of any one of aspects 36 to 41, wherein the capsid comprises an amino acid sequence at least 98% identical to SEQ ID NO: 9.

[0546] 43. The viral particle of any one of aspects 36 to 42, wherein the capsid comprises an amino acid sequence identical to SEQ ID NO: 9.

[0547] 44. The viral particle of any one of aspects 36 to 43, wherein the capsid comprises an amino acid sequence identical to SEQ ID NO: 9. 45. A composition comprising a multiplicity of the viral particles of any one of aspects 36 to 44.

[0548] 46. The composition of aspect 45, wherein the composition has:

[0549] (a) a percentage of viral particles containing within their respective capsids the recombinant genome comprising the transgene that is greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90%; (b) a percentage of viral particles comprising full capsids that is greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90%; and / or

[0550] (c) a full: empty capsid ratio that is greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90%.

[0551] 47. The composition of aspect 45 or 46, wherein the composition has a percentage of viral particles comprising full capsids that is greater than 70%.

[0552] 48. The composition of any one of aspects 45 to 47, wherein the composition has:

[0553] (a) a percentage of viral particles not containing within their respective capsids the recombinant genome comprising the transgene that is less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10%;

[0554] (b) a percentage of viral particles not comprising full capsids that is less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10%; and / or

[0555] (c) an empty:full capsid ratio that is less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10%.

[0556] 49. The composition of any one of aspects 45 to 48, wherein the composition has a percentage of viral particles not comprising full capsids that is less than 30%.

[0557] 50. The composition of any one of aspects 46 to 49, wherein the percentage, full:empty capsid ratio and / or empty:full capsid ratio is measured by SV-AUC. 51. The composition of any one of aspects 45 to 50, wherein the composition further comprises:

[0558] (a) sodium chloride;

[0559] (b) potassium chloride;

[0560] (c) calcium chloride dihydrate;

[0561] (d) magnesium chloride hexahydrate;

[0562] (e) sodium acetate trihydrate;

[0563] (f) sodium citrate dihydrate;

[0564] (g) sodium hydroxide;

[0565] (h) hydrochloric acid; and / or

[0566] (i) polysorbate 20.

[0567] 52. The composition of aspect 51, wherein the composition comprises:

[0568] (a) equal to or greater than 2.5 x 1011vg / ml of the viral particles;

[0569] (b) between 6 and 7 mg / ml sodium chloride;

[0570] (c) between 0.5 and 1 mg / ml potassium chloride;

[0571] (d) between 0.4 and 0.6 mg / ml calcium chloride dihydrate;

[0572] (e) between 0.2 and 0.4 mg / ml magnesium chloride hexahydrate;

[0573] (f) between 3.5 and 4.5 mg / ml sodium acetate trihydrate;

[0574] (g) between 1.5 and 2 mg / ml sodium citrate dihydrate; and / or

[0575] (h) between 0.01 and 0.02% polysorbate 20 (v / v).

[0576] 53. The composition of aspect 51 or aspect 52, wherein the composition comprises:

[0577] (a) equal to or greater than 2.5 x 1011vg / ml of the viral particles;

[0578] (b) between 6 and 7 mg / ml sodium chloride;

[0579] (c) between 0.5 and 1 mg / ml potassium chloride;

[0580] (d) between 0.4 and 0.6 mg / ml calcium chloride dihydrate;

[0581] (e) between 0.2 and 0.4 mg / ml magnesium chloride hexahydrate;

[0582] (f) between 3.5 and 4.5 mg / ml sodium acetate trihydrate;

[0583] (g) between 1.5 and 2 mg / ml sodium citrate dihydrate; and

[0584] (h) between 0.01 and 0.02% polysorbate 20 (v / v). 54. The composition of any one of aspects 45 to 53, wherein the composition has been produced by a method comprising an anion exchange chromatography step.

[0585] 55. The composition of any one of aspects 45 to 54, wherein the composition has been produced by a method which does not comprise a cation exchange chromatography step.

[0586] 56. A method for producing a composition comprising a multiplicity of AAV particles, wherein the method comprises a step of performing anion exchange chromatography on a sample comprising the AAV particles.

[0587] 57. The method of aspect 56, wherein the method is a method for producing a composition comprising a multiplicity of AAV particles having a full: empty capsid ratio that is greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90%.

[0588] 58. The method of aspect 56 or 57, wherein the step of anion exchange chromatography comprises passing the sample through an anion exchange column comprising quaternary polyethyleneimine.

[0589] 59. The method of any one of aspects 56 to 58, wherein the AAV particles are AAV particles according to any one of aspects 36 to 44.

[0590] 60. The method of any one of aspects 56 to 59, wherein the composition is the composition of any one of aspects 45 to 55.

[0591] 61. A composition comprising a multiplicity of AAV particles obtainable by or obtained by the method of any one of aspects 56 to 60.

[0592] 62. The composition of aspect 61, wherein the composition has: (a) a percentage of viral particles containing within their respective capsids the recombinant genome comprising the transgene that is greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90%; (b) a percentage of viral particles comprising full capsids that is greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90%; and / or

[0593] (c) a full: empty capsid ratio that is greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90%.

[0594] 63. The composition of aspect 61 or aspect 62, wherein the composition has:

[0595] (a) a percentage of viral particles not containing within their respective capsids the recombinant genome comprising the transgene that is less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10%;

[0596] (b) a percentage of viral particles not comprising full capsids that is less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10%; and / or

[0597] (c) an empty:full capsid ratio that is less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10%.

[0598] 64. The composition of aspect 62 or aspect 63, wherein the percentage, full:empty capsid ratio and / or empty:full capsid ratio is measured by SV-AUC.

[0599] 65. The composition of any one of aspects 61 to 64, wherein the composition further comprises:

[0600] (a) sodium chloride;

[0601] (b) potassium chloride;

[0602] (c) calcium chloride dihydrate;

[0603] (d) magnesium chloride hexahydrate;

[0604] (e) sodium acetate trihydrate;

[0605] (f) sodium citrate dihydrate;

[0606] (g) sodium hydroxide;

[0607] (h) hydrochloric acid; and / or

[0608] 80 (i) polysorbate 20.

[0609] 66. The composition of aspect 65, wherein the composition comprises:

[0610] (a) equal to or greater than 2.5 x 1011vg / ml of the viral particles;

[0611] (b) between 6 and 7 mg / ml sodium chloride;

[0612] (c) between 0.5 and 1 mg / ml potassium chloride;

[0613] (d) between 0.4 and 0.6 mg / ml calcium chloride dihydrate;

[0614] (e) between 0.2 and 0.4 mg / ml magnesium chloride hexahydrate;

[0615] (f) between 3.5 and 4.5 mg / ml sodium acetate trihydrate;

[0616] (g) between 1.5 and 2 mg / ml sodium citrate dihydrate; and / or

[0617] (h) between 0.01 and 0.02% polysorbate 20 (v / v).

[0618] 67. A vial comprising a stopper and the composition of any one of aspects 45 to 55 or 61 to 66.

[0619] 68. The vial of aspect 67, wherein the stopper is a bromo-butyl stopper or a chloro-butyl stopper.

[0620] 69. A pharmaceutical composition comprising the polynucleotide of any one of aspects 1 to 35, the viral particle of any one of aspects 36 to 44, or the composition of any one of aspects 45 to 55 or 61 to 66 and a pharmaceutically acceptable excipient.

[0621] 70. The polynucleotide of any one of aspects 1 to 35, the viral particle of any one of aspects 36 to 44, the composition of any one of aspects 45 to 55 or 61 to 66, or the pharmaceutical composition of aspect 69 for use in a method of treatment in a subject, wherein the method comprises administering the polynucleotide, the viral particle, the composition or the pharmaceutical composition to the eye.

[0622] 71. A method of treatment in a subject, the method comprising administering the polynucleotide of any one of aspects 1 to 35, the viral particle of any one of aspects 36 to 44, the composition of any one of aspects 45 to 55 or 61 to 66, or the pharmaceutical composition of aspect 69 to the eye.

[0623] 81 72. The polynucleotide, the viral particle, the composition, or the pharmaceutical composition for use according to aspect 70, or the method of aspect 71, wherein administering the polynucleotide, the viral particle, the composition, or the pharmaceutical composition is administration by subretinal injection.

[0624] 73. The polynucleotide of any one of aspects 1 to 35, the viral particle of any one of aspects 36 to 44, the composition of any one of aspects 45 to 55 or 61 to 66, or the pharmaceutical composition of aspect 69 for use in the manufacture of a medicament for use in a method of treatment.

[0625] 74. The polynucleotide, the viral particle, the composition, the pharmaceutical composition for use or the method of any one of aspects 70 to 73, wherein the method of treatment or the method of treating is a method of treating X-linked retinitis pigmentosa (XLRP).

[0626] 75. The polynucleotide for use, viral particle for use, composition for use, or pharmaceutical composition for use according to aspect 70, or 72 to 74 or the method of aspect 71, 72 or 74, wherein the polynucleotide, viral particle, composition or pharmaceutical composition is administered at a dose of 5 x 1010, 7.5 x 1010, 3.7 x 1011, 6.8 x 1011, 1 x 1011or 2 x 1011vg / eye.

[0627] 76. The polynucleotide for use, viral particle for use, composition for use, or pharmaceutical composition for use according to aspect 70, or aspect 72 to 75 or the method of aspect 71, 72 or aspect 74 to 75, wherein the method further comprises administering a steroid.

[0628] 77. The polynucleotide for use, viral particle for use, composition for use, or pharmaceutical composition for use according to aspect 76, or the method according to aspect 76, wherein the steroid is a corticosteroid.

[0629] 78. The polynucleotide for use, viral particle for use, composition for use, or pharmaceutical composition for use according to aspect 76 or aspect 77, or the method according to aspect 76 or aspect 77, wherein the steroid is prednisolone or prednisone.

[0630] 82 79. The polynucleotide for use, viral particle for use, composition for use, or pharmaceutical composition for use according to any one of aspects 76 to 78, or the method according to any one of aspects 76 to 78, wherein the steroid is administered prior to, at the same time as, and / or after the polynucleotide, the viral particle, the composition or the pharmaceutical composition.

[0631] 80. The polynucleotide for use, viral particle for use, composition for use, pharmaceutical composition for use or method according to any one of aspects 70 to 79, wherein the treatment improves visual function of the treated eye of the subject compared to an untreated eye.

[0632] 81. The polynucleotide for use, viral particle for use, composition for use, pharmaceutical composition for use, or method according to 80, wherein the visual function is assessed by measurement of low luminance visual acuity (LLVA) or by performance in a mobility course such as the Ora-VNC™.

[0633] 82. The polynucleotide for use, viral particle for use, composition for use, pharmaceutical composition for use or method according to aspect 80 or aspect 81, wherein the treatment improves the performance of the treated eye of the subject in LLVA by at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 15, at least 16, at least 18, at least 20, at least 22, or at least 24 letters.

[0634] 83. The polynucleotide for use, viral particle for use, composition for use, pharmaceutical composition for use or method according to any one of aspects 80 to 82, wherein the treatment improves the performance of the treated eye of the subject in LLVA by between 10 and 14 letters, or by at least 15 letters.

[0635] 84. The polynucleotide for use, viral particle for use, composition for use, pharmaceutical composition for use or method according to any one of aspects 70 to 83, wherein the treatment improves visual function of the treated eye of the subject to a greater extent than treatment with a corresponding composition having a lower percentage of viral particles comprising full capsids and / or a lower full: empty capsid ratio.

[0636] 83 85. The polynucleotide for use, viral particle for use, composition for use, pharmaceutical composition for use or method according to any one of aspects 70 to 84, wherein the treatment improves the performance of the treated eye of the subject in LLVA by at least 2, at least 4, at least 6, at least 8, or at least 10 letters more than treatment with a corresponding composition having a lower percentage of viral particles comprising full capsids and / or a lower full: empty ratio.

[0637] 86. The polynucleotide for use, viral particle for use, composition for use, pharmaceutical composition for use, or method according to any one of aspects 70 to 85, wherein the treatment improves retinal sensitivity of the treated eye of the subject compared to an untreated eye.

[0638] 87. The polynucleotide for use, viral particle for use, composition for use, pharmaceutical composition for use, or method according to aspect 86, wherein the retinal sensitivity is measured by microperimetry, such as mean sensitivity microperimetry.

[0639] 88. The polynucleotide for use, viral particle for use, composition for use, pharmaceutical composition for use, or method according to aspect 86 or 87, wherein the treatment increases the mean retinal sensitivity of the treated eye of the subject by at least 0.5 dB, at least 1.0 dB, at least 1.5 dB, at least 2.0 dB, or by at least 2.5 dB.

[0640] 89. The polynucleotide for use, viral particle for use, composition for use, pharmaceutical composition for use, or method according to any one of aspects 86 to 88, wherein the treatment increases the mean retinal sensitivity of the treated eye of the subject by at least 2.0 dB.

[0641] 90. The polynucleotide for use, viral particle for use, composition for use, pharmaceutical composition for use, or method according to any one of aspects 86 to 89, wherein the treatment increases the retinal sensitivity of the treated eye of the subject by at least 7 dB at at least 5 loci.

[0642] 84 91. The polynucleotide for use, viral particle for use, composition for use, pharmaceutical composition for use, or method according to any one of aspects 70 to 90, wherein the treatment improves retinal sensitivity to a greater extent than treatment with a corresponding composition having a lower percentage of viral particles comprising full capsids and / or a lower full: empty capsid ratio.

[0643] 92. The polynucleotide for use, viral particle for use, composition for use, pharmaceutical composition for use, or method according to aspect 91, wherein the treatment improves microperimetry mean sensitivity of the treated eye of the subject by at least 1dB more than treatment with a corresponding composition having a lower percentage of viral particles comprising full capsids and / or a lower full: empty capsid ratio.

[0644] 93. The polynucleotide for use, viral particle for use, composition for use, pharmaceutical composition for use, or method according to any one of aspects 70 to 92, wherein the treatment results in fewer serious adverse events than treatment with a corresponding composition having a lower percentage of viral particles comprising full capsids and / or a lower full: empty capsid ratio.

[0645] 94. The polynucleotide for use, viral particle for use, composition for use, pharmaceutical composition for use, or method according to any one of aspects 70 to 93, wherein the subject eye to be treated has a baseline LLD of at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, or at least 24 letters.

[0646] 95. The polynucleotide for use, viral particle for use, composition for use, pharmaceutical composition for use, or method according to any one of aspects 70 to 94, wherein the subject eye to be treated has a baseline LLD of at least 20 letters.

[0647] 96. The polynucleotide for use, viral particle for use, composition for use, pharmaceutical composition for use, or method according to any one of aspects 80 to 95, wherein the improvement is seen at least 3, at least 6, at least 9, at least 12, at least 18, or at least 24 months after treatment.

[0648] 85 97. The polynucleotide for use, viral particle for use, composition for use, pharmaceutical composition for use, or method according to any one of aspects 80 to 96, wherein the improvement is seen after at least 12 months.

[0649] 98. The polynucleotide for use, viral particle for use, composition for use, pharmaceutical composition for use, or method according to any one of aspects 80 to 97, wherein the improvement is seen after at least 24 months.

[0650] 99. The polynucleotide of any one of aspects 1 to 35, the viral particle of any one of aspects 36 to 44, the composition of any one of aspects 45 to 55, the pharmaceutical composition of aspect 69, or the polynucleotide, viral particle, composition or pharmaceutical composition for use of any one of aspects 70 to 99, wherein in the transgene:

[0651] (a) at least 75% of the codons encoding alanine residues are GCA or GCC, optionally wherein the remaining codons encoding alanine are GCT;

[0652] (b) at least 50% of the codons encoding cysteine residues are TGC, optionally wherein about 60% of the codons encoding cysteine residues are TGC;

[0653] (c) at least 30% of the codons encoding aspartate residues are GAC, optionally wherein about 50% of the codons encoding aspartate residues are GAC;

[0654] (d) at least 50% of the codons encoding glutamate residues are GAG, optionally wherein about 57% of the codons encoding glutamate residues are GAG;

[0655] (e) at least 50% of the codons encoding phenylalanine residues are TTC, optionally wherein about 63% of the codons encoding phenylalanine are TTC;

[0656] (f) at least 40% of the codons encoding glycine residues are GGC, optionally wherein about 47% of the codons encoding glycine residues are GGC, optionally wherein none of the codons encoding glycine residues are GGT;

[0657] (g) at least 50% of the codons encoding histidine residues are CAC, optionally wherein about 62% of the codons encoding histidine residues are CAC;

[0658] (h) at least 40% of the codons encoding isoleucine residues are ATC, optionally wherein about 66% of the codons encoding isoleucine residues are ATC, optionally wherein none of the codons encoding isoleucine residues are ATA;

[0659] (i) at least 40% of the codons encoding lysine residues are AAG, optionally wherein about 55% of the codons encoding lysine residues are AAG;

[0660] 86 (j) at least 50% of the codons encoding leucine residues are CTG, optionally wherein all of the codons encoding leucine residues are CTG;

[0661] (k) at least 30% of the codons encoding asparagine residues are AAC, optionally wherein about 54% of the codons encoding asparagine residues are AAC;

[0662] (l) at least 45% of the codons encoding proline residues are CCA, optionally wherein about 48% of the codons encoding proline residues are CCA, optionally wherein none of the codons encoding proline residues are CCG;

[0663] (m) at least 60% of the codons encoding glutamine residues are CAG, optionally wherein all of the codons encoding glutamine residues are CAG;

[0664] (n) at least 50% of the codons encoding arginine residues are CGG, CGA, or CGC, optionally wherein none of the codons encoding arginine residues are CGT;

[0665] (o) at least 60% of the codons encoding serine residues are AGT, AGC, or TCC and / or less than 20% of the codons encoding serine residues are TCA, optionally wherein none of the codons encoding serine residues are TCG;

[0666] (p) at least 20% of the codons encoding threonine residues are ACC, optionally wherein about 40% of the codons encoding threonine residues are ACC, optionally wherein none of the codons encoding threonine residues are ACG;

[0667] (q) at least 60% of the codons encoding valine residues are GTG or GTC, optionally wherein none of the codons encoding valine residues are GTA or GTT; and

[0668] (r) at least 30% of the codons encoding tyrosine residues are TAC, optionally wherein about 50% of the codons encoding tyrosine residues are TAC.

Claims

CLAIMS1. An AAV particle comprising a recombinant genome comprising a capsid and a polynucleotide comprising in 5’ to 3’ order: a 5’ ITR, an hGRKl promoter comprising a nucleotide sequence identical to SEQ ID NO: 17, an SV40 late intron comprising a nucleotide sequence identical to SEQ ID NO: 12, a transgene encoding an RPGR polypeptide comprising an amino acid sequence identical to SEQ ID NO: 11, an SV40 late polyA comprising a nucleotide sequence identical to SEQ ID NO: 13, and a 3’ ITR, wherein in the transgene:(a) at least 75% of the codons encoding alanine residues are GCA or GCC, optionally wherein the remaining codons encoding alanine are GCT;(b) at least 50% of the codons encoding cysteine residues are TGC, optionally wherein about 60% of the codons encoding cysteine residues are TGC;(c) at least 30% of the codons encoding aspartate residues are GAC, optionally wherein about 50% of the codons encoding aspartate residues are GAC;(d) at least 50% of the codons encoding glutamate residues are GAG, optionally wherein about 57% of the codons encoding glutamate residues are GAG;(e) at least 50% of the codons encoding phenylalanine residues are TTC, optionally wherein about 63% of the codons encoding phenylalanine are TTC;(f) at least 40% of the codons encoding glycine residues are GGC, optionally wherein about 47% of the codons encoding glycine residues are GGC, optionally wherein none of the codons encoding glycine residues are GGT;(g) at least 50% of the codons encoding histidine residues are CAC, optionally wherein about 62% of the codons encoding histidine residues are CAC;(h) at least 40% of the codons encoding isoleucine residues are ATC, optionally wherein about 66% of the codons encoding isoleucine residues are ATC, optionally wherein none of the codons encoding isoleucine residues are ATA;(i) at least 40% of the codons encoding lysine residues are AAG, optionally wherein about 55% of the codons encoding lysine residues are AAG;(j) at least 50% of the codons encoding leucine residues are CTG, optionally wherein all of the codons encoding leucine residues are CTG;(k) at least 30% of the codons encoding asparagine residues are AAC, optionally wherein about 54% of the codons encoding asparagine residues are AAC;88(l) at least 45% of the codons encoding proline residues are CCA, optionally wherein about 48% of the codons encoding proline residues are CCA, optionally wherein none of the codons encoding proline residues are CCG;(m) at least 60% of the codons encoding glutamine residues are CAG, optionally wherein all of the codons encoding glutamine residues are CAG;(n) at least 50% of the codons encoding arginine residues are CGG, CGA, or CGC, optionally wherein none of the codons encoding arginine residues are CGT;(o) at least 60% of the codons encoding serine residues are AGT, AGC, or TCC and / or less than 20% of the codons encoding serine residues are TCA, optionally wherein none of the codons encoding serine residues are TCG;(p) at least 20% of the codons encoding threonine residues are ACC, optionally wherein about 40% of the codons encoding threonine residues are ACC, optionally wherein none of the codons encoding threonine residues are ACG;(q) at least 60% of the codons encoding valine residues are GTG or GTC, optionally wherein none of the codons encoding valine residues are GTA or GTT; and(r) at least 30% of the codons encoding tyrosine residues are TAC, optionally wherein about 50% of the codons encoding tyrosine residues are TAC.

2. The AAV particle of claim 1, wherein the hGRKl promoter comprises or consists of a sequence identical to SEQ ID NO: 1.

3. The AAV particle of claim 1 or claim 2, wherein:(i) the 5’ ITR comprises or consists of a sequence:(a) at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 3; or (b) that is identical to SEQ ID NO: 3; and / or(ii) the 3’ ITR comprises or consists of a sequence:(a) at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 4; or (b) that is identical to SEQ ID NO: 4.

4. The AAV particle of any one of the preceding claims, wherein the SV40 late intron comprises or consists of a sequence identical to SEQ ID NO: 5.

895. The AAV particle of any one of the preceding claims, wherein the SV40 late poly A comprises or consists of a sequence identical to SEQ ID NO: 6.

6. The AAV particle of any one of the preceding claims, wherein the polynucleotide is:(a) between 4000 and 5000 nucleotides in length;(b) between 4000 and 4500 nucleotides in length;(c) between 4250 and 4500 nucleotides in length;(d) less than 4500 nucleotides in length; or(e) around 4480 nucleotides in length.

7. The AAV particle of any one of the preceding claims, wherein the AAV particle was produced in mammalian cells.

8. The AAV particle of any one of the preceding claims, wherein the capsid is:(i) an AAV2-derived capsid;(ii) not an AAV5 capsid; or(iii) an AAV5 capsid,preferably wherein the capsid is an AAV2-derived capsid.

9. The AAV particle of any one of the preceding claims, wherein the capsid is an AAV2 capsid comprising a substitution of at least one Y residue with an F residue, optionally wherein the AAV2 capsid comprises a Y444F, Y500F, and / or Y730F substitution.

10. The AAV particle of any one of the preceding claims, wherein the capsid comprises or consists of an amino acid sequence:(a) at least 90%, at least 95%, or at least 98% identical to a fragment of at least 600, at least 650, at least 675, or at least 700 amino acids of SEQ ID NO: 9;(b) at least 95% identical to a fragment of at least 650 amino acids of SEQ ID NO: 9; (c) at least 98% identical to a fragment of at least 700 amino acids of SEQ ID NO: 9; (d) at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 9;(e) at least 98% identical to SEQ ID NO: 9; and / or90(f) identical to SEQ ID NO: 9.

11. The AAV particle of any one of the preceding claims, wherein the transgene comprises:(a) more CpGs than a wild-type RPGR transgene;(b) more than 161 CpGs; and / or(c) about 167 CpGs.

12. The AAV particle of any one of the preceding claims, wherein the transgene comprises or consists of a nucleotide sequence at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% identical or identical to SEQ ID NO: 18.

13. The AAV particle of any one of the preceding claims, wherein the transgene comprises or consists of:(a) a nucleotide sequence identical to SEQ ID NO: 2; or(b) a nucleotide sequence at least 99.5% identical to SEQ ID NO: 2, wherein the nucleotide sequence comprises a T at a position corresponding to position 3457, an A at a position corresponding to position 3458 and an A at a position corresponding to position 3459.

14. The AAV particle of any one of the preceding claims, wherein the polynucleotide comprises or consists of a nucleotide sequence:(a) at least 90%, at least 95%, or at least 98% identical to a fragment of at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, or at least 4250 nucleotides of SEQ ID NO: 8;(b) at least 95% identical to a fragment of at least 4250 nucleotides of SEQ ID NO: 8; (c) at least 98% identical to a fragment of at least 4250 nucleotides of SEQ ID NO: 8; (d) at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 8;(e) at least 98% identical to SEQ ID NO: 8; and / or(f) identical to SEQ ID NO: 8.9115. A composition comprising a multiplicity of the AAV particles of any one of the preceding claims.

16. The composition of claim 15, wherein the composition has:(a) a percentage of viral particles containing within their respective capsids the recombinant genome comprising the transgene that is greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90%; (b) a percentage of viral particles comprising full capsids that is greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90%; and / or(c) a full: empty capsid ratio that is greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90%.

17. A pharmaceutical composition comprising the AAV particle of any one of claims 1 to 14, or the composition of claim 15 or claim 16 and a pharmaceutically acceptable excipient.

18. The AAV particle of any one of claims 1 to 14, the composition of claim 15 or claim 16, or the pharmaceutical composition of claim 17 for use in a method of treatment in a subject, wherein the method comprises administering the polynucleotide, the viral particle, the composition or the pharmaceutical composition to the eye, wherein the method of treatment is a method of treating X-linked retinitis pigmentosa (XLRP).

19. The AAV particle for use, composition for use, or pharmaceutical composition for use according to claim 18, wherein the polynucleotide, viral particle, composition or pharmaceutical composition is administered at a dose of 5 x 1010, 7.5 x 1010, 3.7 x 1011, 6.8 x 1011, 1 x 1011or 2 x 1011vg / eye.

20. The AAV particle for use, composition for use, pharmaceutical composition for use or method according to claim 18 or claim 19, wherein the treatment improves the performance of the treated eye of the subject in LLVA by between 10 and 14 letters, or by at least 15 letters.9221. The AAV particle for use, composition for use, pharmaceutical composition for use, or method according to any one of claims 18 to 20, wherein the treatment increases:(a) the mean retinal sensitivity of the treated eye of the subject by at least 2.0 dB; and / or (b) the retinal sensitivity of the treated eye of the subject by at least 7 dB at at least 5 loci.

22. The AAV particle for use, composition for use, pharmaceutical composition for use, or method according to any one of claims 18 to 21, wherein the subject eye to be treated has a baseline LLD of at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22. or at least 24 letters, preferably wherein the subject eye to be treated has a baseline LLD of at least 20 letters.

23. A method for producing a composition comprising a multiplicity of AAV particles, wherein the method comprises a step of performing anion exchange chromatography on a sample comprising the AAV particles, wherein the method is a method for producing a composition comprising a multiplicity of AAV particles having a full: empty capsid ratio that is greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90%.

24. The method of claim 23, wherein:(a) the AAV particles are AAV particles according to any one of claims 1 to 14; and / or (b) the composition is the composition of claim 15 or claim 16.

25. A composition comprising a multiplicity of AAV particles obtainable by or obtained by the method of claim 23 or claim 24.1 / 18Figure 1

Images