SEQUENCE LISTING SUBMISSION VIA EFS-WEB

A computer readable text file, entitled “090400-5012-US-Sequence-Listing” created on or about Aug. 30, 2021, with a file size of about 37,837 bytes contains the sequence listing for this application and is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

X-linked retinitis pigmentosa (XLRP) is a relatively severe and genetically heterogenous inherited retinal degeneration. Approximately 70% of XLRP cases are caused by mutations in the Retinitis Pigmentosa GTPase Regulator (RPGR) gene. The RPGR gene encodes several distinct alternatively—spliced transcripts that are widely expressed. The function of the encoded protein is not well understood, but studies suggest that it plays an important role in cell structures called cilia.

One RPGR isoform contains a unique 3′ region called ORF15, a Gly- and Glu-rich carboxyl terminal domain of 567 amino acids. This version of the RPGR protein, containing exons 1-13 of the RPGR gene and the ORF15 region, is expressed predominantly in photoreceptors in the retina. Mutations in the ORF15 region of RPGR account for about 60% of all XLRP cases.

Several preclinical studies support the use of wild type cDNA of RPGRorf15 to rescue the XLRP disease phenotype. However, poor sequence stability of the wild type sequence poses challenges to maintaining sequence integrity during vector production and suboptimal expression level of the wild type sequence in human photoreceptors are challenges to gene therapy approaches to treat XLRP.

SUMMARY OF THE INVENTION

Disclosed are codon optimized nucleic acid molecules encoding a human retinitis pigmentosa GTPase regulator (RPGR) protein. In one aspect, the disclosure provides a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1 or a nucleic acid comprising a nucleotide sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:1 and which encodes a human RPGR polypeptide having the amino acid sequence of SEQ ID NO:2. In some embodiments, a nucleic acid comprising or consisting of the nucleotide sequence of SEQ ID NO:1 is provided. In related embodiments, the nucleic acid is expressed at a higher level compared with the level of expression of a wild type RPGR nucleic acid sequence (e.g. SEQ ID NO:3) in an otherwise identical cell.

In some aspects, a codon optimized nucleic acid molecule as herein described has a human codon adaptation index that is increased relative to that of the wild type RPGR cDNA (GenBank Accession No. NM 001034853; SEQ ID NO:3). In some embodiments, the codon optimized nucleic acid molecule has a human codon adaptation index of at least about 0.85, at least about 0.88, or at least about 0.89.

In certain embodiments, the nucleic acid contains a higher percentage of G/C nucleotides compared to the percentage of G/C nucleotides in SEQ ID NO:3. In other embodiments, the nucleic acid contains a percentage of G/C nucleotides that is at most about 59%, at most about 58%, or at most about 57%. In some aspects, the average G/C content of the nucleic acid is from about 55% to about 59%, from about 56% to about 58%. In some preferred embodiments, the average G/C content is about 57%.

In other embodiments, the nucleic acid comprises one or more optimized parameters relative to SEQ ID NO:3 selected from removal of negative cis-acting sites including without limitation TATA-boxes and splice sites, and increasing the frequency of optimal codons.

In another embodiment, the nucleic acid is operatively linked to at least one transcription control sequence, preferably a transcription control sequence that is heterologous to the nucleic acid. In some aspects, the transcription control sequence is a cell- or tissue-specific promoter that results in cell-specific expression of the nucleic acid e.g. in photoreceptor cells such as human rod photoreceptor-specific human G-protein coupled receptor rhodopsin kinase 1 (hGRK) promoter or a human interphotoreceptor retinoid-binding protein (IRBP) promoter. In preferred embodiments, the transcription control sequence comprises a human rod photoreceptor-specific human G-protein coupled receptor rhodopsin kinase 1 (hGRK) promoter. In other aspects, the transcription control sequence is a constitutive promoter that results in similar expression level of the nucleic acid in many cell types (e.g. a CAG, CBA, CMV, or PGK promoter). In preferred embodiments, the transcription control sequence comprises a human G protein-coupled receptor kinase (hGRK, also known as Rhodopsin Kinase) promoter as described in Young et al., Investigative Ophthalmology and Visual Science, 44(9):4076-4085 (2003). In a particularly preferred embodiment, the hGRK promoter comprises the sequence of SEQ ID NO:4 or comprises a sequence at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto:

(SEQ ID NO: 4)GGGCCCCAGAAGCCTGGTGGTTGTTTGTCCTTCTCAGGGGAAAAGTGAGGCGGCCCCTTGGAGGAAGGGGCCGGGCAGAATGATCTAATCGGATTCCAAGCAGCTCAGGGGATTGTCTTTTTCTAGCACCTTCTTGCCACTCCTAAGCGTCCTCCGTGACCCCGGCTGGGATTTAGCCTGGTGCTGTGTCAGCCCCGGG

In related embodiments, provided herein is an expression cassette comprising a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1, or a nucleotide sequence at least 90% identical thereto, operably linked to an expression control sequence.

In related embodiments, provided herein is a vector comprising a comprising a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1, or a nucleotide sequence at least 90% identical thereto. In preferred embodiments, the vector is a recombinant adeno-associated (rAAV) expression vector. In some embodiments, the rAAV vector comprises a native capsid (e.g. a capsid of AAV serotype 2 or AAV serotype 5 or AAV serotype 8). In other embodiments, the rAAV vector comprises a capsid that is modified (e.g. comprises one or more peptide insertions and/or one or more amino acid substitutions (e.g. tyrosine to phenylalanine) and/or amino acid insertions or amino acid deletions) relative to a native AAV capsid (e.g. comprising one or more modifications relative to an AAV capsid of serotype 2, 5 or 8).

In another embodiment, provided herein is a host cell comprising a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1, or a nucleotide sequence at least 90% identical thereto. In some aspects, the host cell is a mammalian cell, including without limitation, a CHO cell, an HEK293 cell, a HeLa cell, a BHK21 cell, a Vero cell or a V27 cell. In related aspects, the host cell is selected from a CHO cell, an HEK293 cell, an HEK293T cell, a HeLa cell, a BHK21 cell and a Vero cell. In other aspects, the host cell is a photoreceptor cell (e.g. rods; cones), a retinal ganglion cell (RGC), a glial cell (e.g. a Müller glial cell, a microglial cell), a bipolar cell, an amacrine cell, a horizontal cell, or a retinal pigmented epithelium (RPE) cell. In related embodiments, the disclosure provides a method of increasing expression of a polypeptide of SEQ ID NO: 2 comprising culturing the host cell under conditions whereby a polypeptide of SEQ ID NO: 2 is expressed by the nucleic acid molecule, wherein the expression of the polypeptide is increased relative to a host cell cultured under the same conditions comprising a reference nucleic acid comprising the nucleotide sequence of SEQ ID NO:3 (comparator sequence).

In another embodiment, the disclosure provides a method of increasing expression of a polypeptide of SEQ ID NO: 2 in a human subject comprising administering to the subject an isolated nucleic acid molecule comprising a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:1 and which encodes a polypeptide having the amino acid sequence of SEQ ID NO:2 or a vector comprising such a nucleotide sequence, wherein the expression of the polypeptide is increased relative to a reference nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:3 (comparator sequence) or a vector comprising the reference nucleic acid molecule.

In some embodiments, the disclosure provides a method of treating an ocular disorder associated with insufficient RGRP ORF15 activity in a human subject comprising administering to the subject a nucleic acid molecule or a vector disclosed herein. In some embodiments, the retinal disorder is X-linked retinitis pigmentosa.

