Field

The invention relates to RNA editing gene therapy vectors for treating or preventing retinal degeneration.

Background

Programmable RNA base editing has been proposed as a therapeutic approach for the correction of point mutations in the transcriptome. The CRISPR-Cas13 system has been harnessed for RNA editing by pairing the programmable RNA binding functions of nucl ease-deactivated Cas13 (dCas13) enzymes with a deaminase domain to mediate base conversions in RNA (Cox et al. (2017) Science 358(6366): 1019; Kannan et al. (2022) Nat. Biotechnol. 40(2): 194). The adenosine deaminases acting on RNA (ADAR) enzymes are endogenously expressed in human cells and mediates the common adenosine-inosine post- transcriptional modification in RNA transcripts. Inosine is recognized as guanine in cellular processes such as translation and splicing, and therefore can be used to install A>G edits and correct G>A mutations. Cas13b-based RNA editing systems paired with the ADAR deaminase domain (dCas13b-ADAR _DD ) have demonstrated robust activity in vitro. However their potential for in vivo correction of G>A mutations has not yet been shown.

Inherited retinal diseases (IRDs) are the most common cause of irreversible sight loss in people of working age. Pathogenic variants in the gene USH2A are a leading cause of IRDs (Pontikos), which is associated with autosomal recessive non-syndromic retinitis pigmentosa, as well as Usher syndrome type II, characterized by congenital sensorineural hearing loss and retinitis pigmentosa. Adeno-associated virus (AAV)-mediated gene replacement therapy is now approved for RPE65-associated Lebers Congenital Amaurosis and is being investigated for several other IRDs in clinical trials. The large (15.6kb) coding sequence of USH2A that encodes the usherin protein greatly exceeds the ~4.7kb capacity of a single AAV vector however, and alternative treatment strategies are required.

Summary

The inventors have for the first time demonstrated RNA editing in vivo in the retina and in photoreceptor cells. They have also demonstrated for the first time Cas13-ADAR activity in vivo; and more specifically, Cas13-ADAR activity in the retina and in photoreceptor cells. They have also demonstrated for the first time in vivo activity ofCas13-ADAR from AAV vectors. Accordingly, in a first aspect the invention provides a method of treating or preventing retinal degeneration in a subject in need thereof, the method comprising administering to the subject (i) an adeno-associated virus (AAV) vector that encodes a dCas 13b- ADAR _DD fusion protein comprising nucl ease-deactivated Cas13 (dCas13) and the deaminase domain of an adenosine deaminase acting on RNA (ADAR _DD ); and (ii) an adeno-associated virus (AAV) vector that encodes a guide RNA; wherein the dCas13b- ADAR and the guide RNA are encoded on the same vector, or on two different vectors, administered to the subject; wherein the guide RNA binds to a target RNA expressed in the retina; and wherein there is a nucleotide sequence mismatch between the guide RNA and the target RNA at a site corresponding to a pathogenic mutation or variant.

In an further aspect, the invention provides an adeno-associated virus (AAV) vector, or a pair of adeno-associated virus (AAV) vectors, wherein (I) the AAV vector encodes a dCas13b-ADAR _DD fusion protein and a guide RNA, wherein the guide RNA hybridises to a target RNA expressed in the retina; and wherein there is a nucleotide sequence mismatch between the guide RNA and the target RNA at a site corresponding to a pathogenic mutation or variant; or (II) one of the pair of AAV vectors encodes a dCas 13b- ADAR _DD fusion protein and the other AAV vector of the pair encodes a guide RNA; wherein the guide RNA hybridises to a target RNA expressed in the retina; and wherein there is a nucleotide sequence mismatch between the guide RNA and the target RNA at a site corresponding to a pathogenic mutation or variant.

In a further aspect, the invention provides:

- The vector or vectors for use in a method of treatment;

- The vector or vectors for use in a method of treating or preventing retinal degeneration;

- The use of the vector or vectors in the manufacture of a medicament; and

- The use of the vector or vectors in the manufacture of a medicament for treating or preventing retinal degeneration.

In a further aspect, the invention provides a pharmaceutical composition or a kit or panel of two pharmaceutical compositions, comprising the vector or the pair of vectors described above, and optionally at least one pharmaceutically acceptable diluent, carrier, or preservative; wherein the pair of vectors are combined in the same pharmaceutical composition, or are separated in the two pharmaceutical compositions of the kit or panel.

In a further aspect, the invention provides a host cell comprising the or a vector as described above. The invention will now be described in more detail, by way of example and not limitation, and by reference to the accompanying drawings. Many equivalent modifications and variations will be apparent, to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the disclosure set forth are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the scope of the invention which is defined by the claims. All documents cited herein, whether supra or infra, are expressly incorporated by reference in their entirety.

The present disclosure includes the combination of the aspects and features described except where such a combination is clearly impermissible or is stated to be expressly avoided. As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a vector” includes two or more such entities. In general, the term “comprising” is intended to mean including but not limited to. In some embodiments of the invention, the word “comprising” is replaced with the phrase “consisting of" or the phrase “consisting essentially of". The term “consisting of" is intended to be limiting. The term “consisting essentially of" should be understood to mean that the sequence comprises no additional sequence units or elements that materially affect the function of the sequence element.

Section headings are used herein for convenience only and are not to be construed as limiting in any way.

Description of the Figures

Fig 1. Schematic demonstrating the REPAIR system with dPspCas13b-ADAR fusions recruited by a guide RNA with a direct repeat. Note the A-C mismatch at the target adenosine to promote editing at the target site. gRNA = guide RNA; dPspCas13b = deactivatedCas13b orthologue derived fromPrevotella sp. P5-125.

Fig 2. In vitro optimisation of dCas13b-ADAR _DD editing of USH2A

A. Schematic of triple transfection dual luciferase assay in HEK293T cells for screening of RNA editing. A-I editing of a premature stop codon upstream of firefly results in restoration of firefly activity. Renilla acts as a normalization control. B. Representative sanger sequencing chromatograms following editing. C. Schematic diagrams of dPspCas13b-ADAR _DD and dCas13bt-ADAR _DD plasmid constructs, and of gRNA with mismatch distance between A-C mismatch at target adenosine and gRNA scaffold. D-E. Screening of gRNAs against the USH2A p.W3955X and Ush2a p.W3947X targets with dPspCas13b(del)-E488Q. F-G. Screening of 30nt and 50nt gRNAs against Ush2a p.W3947X target with dCas13bt-E488Q constructs. H-L Comparison of dPspCas13b- ADAR _DD constructs. Two-way ANOVA, Tukey’s multiple comparison testing. *** p < 0.001, **** p < 0.0001 J. Quantification of editing rates with dPspCas13b-ADAR _DD by decomposition of Sanger chromatograms with MultiEditR. K. Local bystander deamination of adenosines by dPspCas13b-ADAR _DD across the USH2A and Ush2a target sequences, quantified with MultiEditR. Data all mean ± SEM, n = 3.

Fig. 3. Dose response testing of dPspCas13b-ADAR2 _DD constructs

A. Repair efficiencies calculated using hUSH2A-W3955X dual luciferase construct. HEK293T cells were transfected with 50ng of luciferase plasmid, two doses of the dPspCas13bdel-ADAR2DD construct (75ng and 150ng) and increasing doses of a hUSH2A targeting guide plasmid. B. Representative light microscope images ( 10x magnification) of confluent HEK293T cells 48hrs post-transfection prior to lysis for luciferase assay. Toxicity is seen at the highest dose of plasmids (150ng Cas13b, 300ng guide RNA, 50ng luciferase plasmid) with reduced confluency of cells likely due to cell death. Scale bar, 100μm. Data mean ± SEM, n = 3

Fig. 4. Correlation between RNA editing with dPspCas13b-ADAR2 _DD measured by luciferase assay and sanger sequencing

On-target editing rates as measured using the luciferase assay (x axis) decomposition of sanger sequencing (y axis) from adjacent samples transfected identically in the same plate are plotted. Results are from samples transfected with different guides and different editing constructs. A curve fitted with local regression (loess) is plotted with 95% confidence intervals. A positive relationship between editing rates detected by each method is observed (Pearson correlation R ² = 0.78). Editing rates are observed as higher by sanger sequencing for a given sample, compared to editing rate detected with the luciferase assay. Editing rates determined by sanger sequencing appear to approach a ceiling of maximum editing.

Fig. 5. Usherin expression is not detected in retina of Ush2a-W3947X mouse

A. Sanger sequencing chromatograms of gDNA of offspring of F2 heterozygous intercrosses demonstrating homozygous Us2ha c.11840 G>A (p.W3947X) and wildtype offspring. B. Western blot of protein lysates from the neural retina and eye cup (comprising RPE, sclera and choroid), each lane representing an eye of one animal. No usherin expression is seen in Ush2a-W3947X homozygotes, with appropriately sized bands (predicted ~590kDA) in wildtype animals. Expression is specific to the neural retina. Vinculin used as a loading control. C. Immunofluorescence images of retinal sections from 11 wk old animals using a 40x overview (scale = 50μm) of the full thickness of the retina and a 63x image (scale = 30μm) of the outer retina (inset). A schematic demonstrates the cellular localisation usherin at the connecting cilium at the tips of rootletin staining demarcating the inner segment. No usherin expression is seen in the Ush2a-W3947X homozygous mouse, while punctate usherin staining is observed in the wildtype mouse.

Fig. 6. Generation and genotyping of Ush2a-W3947X mouse

A. Locus map of Ush2a exon 60. The gRNA sequence (blue) designed to generate a double stranded break for subsequent homology directed repair from the single stranded oligo donor nucleotide (ssODN) template sequence containing the c.l 18640G>A (p.W3947X) mutation is annotated. This mutation abolishes an Avail restriction site in the WT sequence. Primers and probes for allelic discrimination genotyping assay are annotated. B. Sanger sequencing of founder F ₁ heterozgyote performed by MRC Harwell. C. ddPCR copy counting assay of founder F1 offspring (labelled F3-F6) showing a single copy of the mutant allele and no more than two copies of Ush2a

Fig. 7. In vivo RNA editing of photoreceptors with AAV-dCas13b-ADAR

A. Construct maps for each of the three injected AAV vectors. The dual vector approach used dPspCas13b-ADAR _DD (E488Q) driven by either an EFS or RK promoter, together with a gRNA vector also expressing GFP. The all-in-one vector approach included dCas13bt3-ADAR _DD (E488Q) together with a gRNA. B. Experimental outline for in vivo experiments. C. En face in vivo CSLO retinal imaging. Representative infrared reflectance image showing optic nerve head centrally. Blue autofluorescence imaging demonstrates exogenous GFP expression in injected retinas, with no expression seen in PBS injected retinas. D. Electrophoresis of RT-PCR products of Cas13-ADAR transgenes from injected retinas. E. On-target A>G editing rates for each construct. One-way ANOVA relative to PBS control with Dunnet’s multiple comparisons testing (* p = 0.0124, *** p < 0.0001). Data shown as mean ± SEM. F. Immunohistochemistry of retinal sections from eyes injected with the dual RK-dPspCas13-E488Q vectors. Rootletin staining defines the inner segment to the connecting cilium, with Usherin present at the tips. Weak restoration of Usherin is seen in eyes injected with the targeting gRNA in GFP-positive transduced areas of retina.

Fig. 8. Testing of minimal elements for AAV-dPspCas13b-ADAR constructs Comparison of RNA editing rates when using minimal promoters and poly A elements using luciferase assay in transfect HEK293T. A. Comparison of RNA Pol II promoters driving dPspCas13-E488Q. Two-way ANOVA with promoter and gRNA as factors: F(2, 12) = 6.89, p = 0.01 for effect of promoter; F (2, 12) = 0.058, p = 0.94 for interaction between guide and promoter. No significant difference on Tukey’s multiple comparison testing. B. Comparison polyA signals for dPspCas13-E488Q demonstrates efficient editing with the snRPl polyA. Two-way ANOVA with polyA and gRNA as factors: F(1, 8) = 1.67, p = 0.23 for effect of polyA; F (1, 8) = 0.0001, p = 0.99 for interaction between guide and polyA. C. Comparison of RNA Pol III promoters driving gRNA demonstrates loss of editing efficiency with a tRNA promoter. Two-way ANOVA with promoter and gRNA as factors: F(1, 8) = 27.64, p = 0.008 for effect of promoter; F (1, 8) = 0.029, p = 0.87 for interaction between promoter and guide. **p = 0.009 and *p = 0.014, Tukey’s multiple comparison test. All data shown as mean ± SEM, n = 3. Data from the CMV- IE.dPspCas13b-del-ADAR _DD (E488Q) with a hU6 driven gRNA construct (blue bars) used as a reference and displayed in A, B and C. Plasmid maps displayed over graph, with variable region in green. D. Comparison of dual vector plasmid constructs with minimal elements for AAV-packaging with original (reference) plasmids used in gRNA screening shown in plasmid maps. Equivalent editing rates were seen between constructs for both guides, without loss of editing efficiency from the weaker EFS promoter and sNRPl terminator (Two-way ANOVA between targeting guides with construct and gRNA as factors, F(1,8)=0.18, p=0.68 for effect of construct). E. Western blot of protein lysates from cells in 12-well plates transfected with increasing doses of EFS-dPspCas13bdel- ADAR2DD-SNRP1. Transgene expression detected with anti -FLAG staining to c-terminal flag tag with the appropriate predicted size (~160kDA, red). A c-terminal FLAG tagged recombinant bacterial alkaline phosphatase (rB AP) protein (~49kDA) was used as a positive control, with β-actin as a protein loading control (green). F. Immunocytochemistry following transfection with the AAV-EFS-dPspCas13bdel-ADAR2dd-sNRPl construct. dPspCas13bdel-ADAR2DD expression detected by anti-FLAG staining is observed in the cytoplasmic compartment, as expected due to the fused HIV-derived nuclear export signal.

Fig 9. Transcript analysis of dPspCas13-E488Q injected retinas

A. Vector map of AAV-EFS-dPspCas13b-E488Q showing locations of primer pairs. B. Analysis using RT-PCR with primer pairs targeting the transcriptional start site (TSS) of the EFS promoter and the beginning of the polyA tail, with further pairs walked in along the transgene. No deletions were observed in products amplified from cDNA (C) compared to plasmid DNA (P). Amplification was not observed in the no-template control (NTC, N). C. Sanger sequencing coverage plot of the Fl + R1 RT-PCR product with full coverage and no errors detected. D. Vector map of AAV-RK-dPspCas13b-E488Q showing locations of primer pairs. E. Similar RT-PCR analysis as in B, of the AAV-RK-dPspCas13b-E488Q vector. F. Sanger sequencing coverage plot of the Fl + R1 RT-PCR product with full coverage and no errors detected.

Fig. 10. Off-target editing of Ush2a with AAV-dCas13b-ADAR

A. Off-target analysis by deep sequencing, demonstrating A>G editing rate at every local adenosine in the Ush2a exon 60 amplicon for each vector (n = 6 retinas per vector, PBS n = 8). Significant editing sites outlined in black (adjusted p<0.01, PBS injected eyes as controls). gRNA binding region demonstrated from A ₂₈ to A ₄₀ . B. Editing rates of all significant off-target sites plotted on a log-scale. Mean background rate of A>G calls in sequencing data in transcripts in PBS injected eyes shown as dotted line. C. Editing rate (A>G) of Ush2a transcripts demonstrating proportion of all amplicon reads corrected at the target base, and proportion of reads corrected at target base without any other off-target editing in the transcript. All data shown as mean ± SEM. n.d. = not detected. Significant editing sites determined with Fisher’s exact test (two-tailed) with multiple comparison testing using a false discovery rate (FDR) controlled at 5% with the Benjamini -Hochberg method.

Fig. 11. Figure 1 | Effect of injection on retinal thickness and structure

A. Photoreceptor layer thickness (PRL) at 4-weeks post subretinal injection measured at the superior retina (at injection site) and inferior retina (opposite injection site). Superior retinal thinning was observed at the injection site of all vectors except RK-Cas13bt3, but not those injected with a buffer-only control. Doses administered were 1E+9gc/eye of Cas13 vector and 1E+9gc/eye of gRNA vector for the EFS-Psp group, 1E+9gc/eye of Cas13 vector and 5E+8gc/eye of gRNA vector for the RK-Psp group, and 1E+9gc/eye of single vector Cas13-gRNA for the RK-Cas13bt3 group. Paired two way ANOVA for effect of retinal location (F(1, 68)=92.35, P<0.0001) with Sidak’s multiple comparison testing. Mean ± SEM. B. CSLO images acquired with blue autofluorescence (BAF) imaging with a bandpass filter (BP), infrared reflectance (IR) and OCT for each condition. BAF imaging shows robust GFP expression in the superior retina from GFP constructs. The RK-Cas13bt constructs did not contain a GFP expression cassette. In eyes with retinal thinning, this was seen principally in the outer retina, and associated with loss of the hyper-reflective ellipsoid zone and external limiting membrane bands. BAF images acquired at 90 detector sensitivity. Scale bars = 200 μm.

Fig. 12. Immunofluorescence images of retinal sections injected with EFS- dPspCas13b -E488Q and RK-dCas13bt3-E488Q AAV vectors

A. Representative retinal immunofluorescence images from mice injected with EFS- dPspCas13b-E488Q vectors, at 40x (scale = 30μm) with inset 63x images focused on the distal inner segment (scale = 5μm). Endogenous GFP fluorescence from the gRNA-GFP vector is seen at the injection site in treated animals. Clear usherin labelling is observed in the wildtype control but not in the AAV injected animals. B. Representative retinal immunofluorescence images from mice injected with RK-dCas13bt3-E488Q vectors. Usherin restoration is not observed.

Description of the Sequences

SEQ ID NO: 1 set forth the RNA sequence of Homo sapiens usherin (USH2A), mRNA transcript variant 2.

SEQ ID NO: 2 sets forth the RNA sequence of Homo sapiens usherin (USH2A), transcript variant 2, mRNA Exon 61

SEQ ID NO: 3 sets forth the DNA sequence of dPspCas13(A984-1090).

SEQ ID NO: 4 sets forth a DNA sequence for Cas13btl .

SEQ ID NOs: 5 sets forth a DNA sequence for Cas13bt3.

SEQ ID NOs: 6 sets forth a DNA sequence for Cas13bt5.

SEQ ID NO: 7 sets forth a DNA sequence of ADAR _DD .

SEQ ID NO: 8 sets forth the DNA sequence of ADAR _DD (E488Q).

SEQ ID NO: 9 sets forth the DNA sequence of ADAR _DD (E488Q, T375G).

SEQ ID NO: 10 sets forth the amino acid sequence of ADAR _DD of wild type hADAR2.

SEQ ID NO: 11 sets forth the sequence of the human rhodopsin kinase promoter (GRK1) (-112/+87).

SEQ ID NO: 12 sets forth the DNA sequence of a hU6 promoter.

SEQ ID NO: 13 sets forth the DNA sequence of a dual soluble neuropilin- 1 (sNRPl) polyadenylation signal.

SEQ ID NO: 14 sets forth the amino acid sequence of dPspCas13b-ADAR _DD (E488Q)- FLAG.

SEQ ID NO: 15 sets forth the DNA sequence of a HIV nuclear export signal (NES). SEQ ID NO: 16 sets forth the DNA Sequence Full RK-PspCas13-ADAR-E488Q vector, including AAV2 ITRs.

SEQ ID NO: 17 sets forth the DNA sequence of the full U6-gRNA-CAG-GFP-WPRE cloning vector insert (without spacer cloned in).

SEQ ID NO: 18 sets forth a SV40 PolyA DNA Sequence.

SEQ ID NO: 19 sets forth a AAV2/8-Y733F RepCap Gene Sequence.

SEQ ID NO: 20 sets forth a guide RNA spacer sequence.

SEQ ID NO: 21 sets forth the amino acid sequence of dPstCas13 (A984-1090).

SEQ ID NO: 22 sets forth the amino acid sequence of ADAR _DD (E488Q).

SEQ ID NO: 23 sets forth the amino acid sequence of ADAR _DD (E488Q, T375G).

SEQ ID NO: 24 sets forth the DNA sequence of the RK-PspCas13-ADAR _DD (E488Q) vector insert.

SEQ ID NO: 25 sets forth the DNA sequence of the U6-gRNA vector insert.

SEQ ID NO: 26 sets forth the AAV2 5’ITR sequence.

SEQ ID NO: 27 sets forth the AAV2 3’ITR sequence.

SEQ ID NO: 28 sets forth the sequence of the human rhodopsin promoter (RHOp).

SEQ ID NO: 29 sets forth the chicken beta-actin promoter exon-intron-exon sequence

SEQ ID NO: 30 sets forth the woodchuck hepatitis post-transcriptional regulatory element (WPRE) sequence.

SEQ ID NO: 31 sets forth the bovine growth hormone polyadenylation tail sequence.

SEQ ID NO: 32 sets forth a guide RNA sequence.

SEQ ID NO: 33 sets forth the DNA Sequence of a vector for expressing a guide RNA.

SEQ ID NO: 34 sets forth the amino acid sequences of a HIV NES.

SEQ ID NO: 35 sets forth the amino acid sequences of a MAPK NES

SEQ ID NO: 36 sets forth the amino acid sequences of a PKI-alpha NES.

SEQ ID NO: 37 sets forth the amino acid sequences of a FAK NES2.

SEQ ID NO: 38 sets forth the amino acid sequence of dPspCas13b-ADAR _DD (E488Q).

SEQ ID NO: 39 sets forth the amino acid sequence of dPspCas13b-ADAR _DD (E488Q, T375G).

SEQ ID NO: 40 sets forth the DNA sequence of the RK-PspCas13-ADAR _DD (E488Q, T375G) vector insert.

SEQ ID NO: 41 sets forth the sequence of AAV-hU6-gRNA-CAG-GFP, as described in Example 5. SEQ ID NO: 42 sets forth the coding sequence (CDS) of the RNA sequence of reference Homo sapiens usherin (USH2A), mRNA transcript variant 2 that encodes the Usherin protein.

Detailed Description

Vectors

The invention relates to vectors and gene therapy vectors. A gene therapy vector is any vector suitable for use in gene therapy, i.e. any vector suitable for the therapeutic delivery of nucleic acid polymers into target cells. In the present case, the gene therapy vectors are RNA editing vectors. The vectors provide RNA editing machinery and a suitable guide RNA to target a specific edit in RNA expressed in the retina. One or two vectors may be used depending on the size of the inserts and the carrying capacity of the vectors.

Adeno-associated virus (AAV) vectors

The vector may comprise a genome from a naturally derived serotype, isolate or clade of AAV or a derivative or one or more functional units thereof. An AAV genome is a polynucleotide sequence which encodes one or more functions needed for production of an AAV viral particle. Naturally occurring AAV viruses are replication-deficient and rely on the provision of helper functions in trans for completion of a replication and packaging cycle. Accordingly, the AAV genome of the vector of the invention is typically replicationdeficient.

The AAV genome may be in single-stranded form, either positive or negativesense, or in double-stranded form. The use of a double-stranded form allows bypass of the DNA replication step in the target cell and so can accelerate transgene expression.

In general, for therapeutic purposes, the only sequences required in cis, in addition to the therapeutic gene is at least one inverted terminal repeat sequence (ITR). In naturally derived AAV, the ITR sequence(s) act in cis to provide a functional origin of replication, and allows for integration and excision of the vector from the genome of a cell. The natural AAV genome also comprises packaging genes, such as rep and/or cap genes which encode packaging functions for an AAV viral particle. The rep gene encodes one or more of the proteins Rep78, Rep68, Rep52 and Rep40 or variants thereof. The cap gene encodes one or more capsid proteins such as VP1, VP2 and VP3 or variants thereof. These proteins make up the capsid of an AAV viral particle. Capsid variants are discussed below. A promoter may be operably linked to each of the packaging genes. Specific examples of such promoters include the p5, pl 9 and p40 promoters (Laughlin et al., 1979, PNAS, 76:5567-5571). For example, the p5 and pl9 promoters are generally used to express the rep gene, while the p40 promoter is generally used to express the cap gene.

In therapeutic AAVs, the cap and/or rep genes may be removed. The removal of the viral genes renders rAAV incapable of actively inserting its genome into the host cell DNA. Instead, the rAAV genomes fuse via the ITRs, forming circular, episomal structures, or insert into pre-existing chromosomal breaks. For viral production, the structural and packaging genes, now removed from the rAAV, are supplied in trans, in the form of a helper plasmid. This is discussed further below. Removal of the cap and/or rep genes provides additional capacity for the insertion of a transgene such as, in the present case, PspCasl3b. Hence, the gene therapy vectors described herein are recombinant viral vectors.

As is known to the skilled person, AAV viruses occurring in nature may be classified according to various biological systems.

Commonly, AAV viruses are referred to in terms of their serotype. A serotype corresponds to a variant subspecies of AAV which, owing to its profile of expression of capsid surface antigens, has a distinctive reactivity that can be used to distinguish it from other variant subspecies. Typically, a virus having a particular AAV serotype does not efficiently cross-react with neutralising antibodies specific for any other AAV serotype. AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11, also recombinant serotypes, such as Rec2 and Rec3, identified from primate brain. In vectors of the invention, the genome may be derived from any suitable AAV serotype, such as AAV2, AAV5, AAV8, AAV9 or serotypes derived from processes such as directed evolution or ancestral derivation, for instance but not limited to AAV-Anc80L65 or AAV-PHP.eB.

The capsid may also be derived from any suitable AAV serotype, such as AAV8. For example, the capsid may have the amino acid sequence of SEQ ID NO: 19 (AAV2/8- Y733F) or may be a variant thereof having at least 80%, or at least 85%, 87%, 90%, 95%, 97%, 98%, or 99% sequence identity therewith. Reviews of AAV serotypes may be found in Choi et al. (Curr Gene Ther. 2005; 5(3); 299-310) and Wu et al (Molecular Therapy. 2006; 14(3), 316-327).

Examples of AAV genome sequences that may in some cases be suitable, or of functional sequence units, including ITR sequences, rep or cap genes and regulatory elements, that may in some cases be suitable, may be derived from the following accession numbers: Adeno-associated virus 1 NC 002077.1, AF063497; Adeno-associated virus 2 NC 001401.2; Adeno-associated virus 3 NC_001729.1; Adeno-associated virus 3B NC 001863; Adeno-associated virus 4 NC 001829; Adeno-associated virus 5 Y18065, AF085716; Adeno-associated virus 6 NC 001862; Avian AAV ATCC VR-865 AY186198, AY629583, NC_004828; Avian AAV strain DA-1 NC_006263, AY629583; Bovine AAV NC_005889, AY388617.

AAV viruses may also be referred to in terms of clades or clones. This refers to the phylogenetic relationship of naturally derived AAV viruses, and typically to a phylogenetic group of AAV viruses which can be traced back to a common ancestor, and includes all descendants thereof. Additionally, AAV viruses may be referred to in terms of a specific isolate, i.e. a genetic isolate of a specific AAV virus found in nature. The term genetic isolate describes a population of AAV viruses which has undergone limited genetic mixing with other naturally occurring AAV viruses, thereby defining a recognisably distinct population at a genetic level.

Examples of clades and isolates of AAV that may similarly be suitable include:

Clade A: AAV1 NC_002077, AF063497, AAV6 NC_001862, Hu. 48 AY530611, Hu 43 AY530606, Hu 44 AY530607, Hu 46 AY530609

Clade B: Hu. 19 AY530584, Hu. 20 AY530586, Hu 23 AY530589, Hu22 AY530588, Hu24 AY530590, Hu21 AY530587, Hu27 AY530592, Hu28 AY530593, Hu 29 AY530594, Hu63 AY530624, Hu64 AY530625, Hul3 AY530578, Hu56 AY530618, Hu57 AY530619, Hu49 AY530612, Hu58 AY530620, Hu34 AY530598, Hu35 AY530599, AAV2 NC .001401, Hu45 AY530608, Hu47 AY530610, Hu51 AY530613, Hu52 AY530614, Hu T41 AY695378, Hu S17 AY695376, Hu T88 AY695375, Hu T71 AY695374, Hu T70 AY695373, Hu T40 AY695372, Hu T32 AY695371, Hu T17 AY695370, Hu LG15 AY695377,

Clade C: Hu9 AY530629, Hu10 AY530576, Hul l AY530577, Hu53 AY530615, Hu55 AY530617, Hu54 AY530616, Hu7 AY530628, Hul8 AY530583, Hul5 AY530580, Hul6 AY530581, Hu25 AY530591, Hu60 AY530622, Ch5 AY243021, Hu3 AY530595, Hui AY530575, Hu4 AY530602 Hu2, AY530585, Hu61 AY530623

Clade D: Rh62 AY530573, Rh48 AY530561, Rh54 AY530567, Rh55 AY530568, Cy2 AY243020, AAV7 AF513851, Rh35 AY243000, Rh37 AY242998, Rh36 AY242999, Cy6 AY243016, Cy4 AY243018, Cy3 AY243019, Cy5 AY243017, Rhl3 AY243013 Clade E: Rh38 AY530558, Hu66 AY530626, Hu42 AY530605, Hu67 AY530627, Hu40 AY530603, Hu41 AY530604, Hu37 AY530600, Rh40 AY530559, Rh2 AY243007, Bbl AY243023, Bb2 AY243022, Rh10 AY243015, Hul7 AY530582, Hu6 AY530621, Rh25 AY530557, Pi2 AY530554, Pil AY530553, Pi3 AY530555, Rh57 AY530569, Rh50 AY530563, Rh49 AY530562, Hu39 AY530601, Rh58 AY530570, Rh61 AY530572, Rh52 AY530565, Rh53 AY530566, Rh51 AY530564, Rh64 AY530574, Rh43 AY530560, AAV8 AF513852, Rh8 AY242997, Rhl AY530556

Clade F: Hul4 (AAV9) AY530579, Hu31 AY530596, Hu32 AY530597, Clonal Isolate AAV5 Y18065, AF085716, AAV 3 NC_001729, AAV 3B NC_001863, AAV4 NC 001829, Rh34 AY243001, Rh33 AY243002, Rh32 AY243003Z

The skilled person can select an appropriate serotype, clade, clone or isolate of AAV for use in the present invention on the basis of their common general knowledge. It should be understood however that the invention also encompasses use of an AAV genome of other serotypes that may not yet have been identified or characterised.

The AAV genome used in the invention may be the full genome of a naturally occurring AAV virus. However, while such a vector may in principle be administered to patients, this will be done rarely in practice. The AAV genome may instead be derivatised for the purpose of administration to patients. Such derivatisation is standard in the art and the present invention encompasses the use of any suitable known derivative of an AAV genome, and derivatives which could be generated by applying techniques known in the art. Derivatisation of the AAV genome and of the AAV capsid (discussed below) are reviewed in Coura and Nardi (Virology Journal, 2007, 4:99), and in Choi et al. and Wu et al., referenced above.

Derivatives of an AAV genome include any truncated or modified forms of an AAV genome which allow for expression of the dCas13b-ADAR _DD fusion protein and/or guide RNA from the vector in vivo in accordance with the present invention. Typically, it is possible to truncate the AAV genome significantly to include minimal viral sequence yet retain the above function. Reducing the size of the AAV genome in this way allows for increased flexibility in incorporating other sequence elements such as regulatory elements within the vector. It may also reduce the possibility of integration of the vector into the host cell genome, reduce the risk of recombination of the vector with wild-type virus, and avoid the triggering of a cellular immune response to viral gene proteins in the target cell.

Typically, a derivative will include at least one inverted terminal repeat sequence (ITR), or two ITRs or more. Typically the vector will have two ITRs, that flank the transgene. In some cases, the ITRs may be derived from AAV genomes having different serotypes, or may be a chimeric or mutant ITR. An example mutant ITR is one having a deletion of a trs (terminal resolution site). This deletion allows for continued replication of the genome to generate a single-stranded genome which contains both coding and complementary sequences i.e. a self-complementary AAV genome. This allows for bypass of DNA replication in the target cell, and so enables accelerated transgene expression.

The one or more ITRs may flank a polynucleotide sequence encoding the dCas13b- ADAR _DD fusion protein and or guide RNA at either end. The inclusion of one or more ITRs may aid concatemer formation of the vector of the invention in the nucleus of a host cell, for example following the conversion of single-stranded vector DNA into doublestranded DNA by the action of host cell DNA polymerases. The formation of such episomal concatemers protects the vector construct during the life of the host cell, thereby allowing for prolonged expression of the transgene in vivo The ITR sequences may, for example, be those of AAV2 having, for example, the sequence of SEQ ID NOs: 26 (5’ITR) and/or 27 (3’UTR) or variants having at least 80% or 85%, or 90%, or 95% or 98% or 99% sequence identity to SEQ ID NOs: 26 or 27, or up to 1, 2, 3, 4 or 5 insertions, deletions, or substitutions in the amino acid sequences of SEQ ID NO: 26 or 27.

In some embodiments, ITR elements may be the only AVV sequences retained in the vector. In some embodiments, one or more rep and/or cap genes or other viral sequences may be retained. Naturally occurring AAV virus integrates with a high frequency at a specific site on human chromosome 19, and shows a negligible frequency of random integration, such that retention of an integrative capacity in the vector may be tolerated in a therapeutic setting.

The invention additionally encompasses the provision of sequences of an AAV genome in a different order and configuration to that of a native AAV genome. The invention also encompasses the replacement of one or more AAV sequences or genes with sequences from another virus or with chimeric genes composed of sequences from more than one virus. Such chimeric genes may be composed of sequences from two or more related viral proteins of different viral species.

Capsid coats, Viral particles, Vesicles, Nanoparticles and Exosomes

A viral vector of the invention may have a capsid coat. Such an encapsidated vector may be referred to as a viral particle. The vectors or particles of the invention include transcapsidated forms wherein an genome or derivative having the ITR(s) or other genome components of one serotype or virus type, for example AAV2, is packaged in the capsid of a different serotype, for example AAV8. This may be referred to as pseudotyping. The vectors or particles of the invention also include mosaic forms wherein a mixture of modified or unmodified capsid proteins from two or more different serotypes makes up the viral coat. The invention encompasses the provision of capsid protein sequences from different serotypes, clades, clones, or isolates within the same vector or viral particle. The vector may be a chimeric, shuffled or capsid modified derivative.

The capsid coat is typically selected to provide one or more desired functionalities for the viral vector, such as increased efficiency of gene delivery, decreased immunogenicity (humoral or cellular), an altered tropism range and/or improved targeting of a particular cell type compared to a viral vector comprising a naturally occurring genome. Increased efficiency of gene delivery may be effected by improved receptor or coreceptor binding at the cell surface, improved internalisation, improved trafficking within the cell and into the nucleus, improved uncoating of the viral particle and improved conversion of a single-stranded genome to double-stranded form. Increased efficiency may also relate to an altered tropism range or targeting of a specific cell population, such that the vector dose is not diluted by administration to tissues where it is not needed.

The capsid may determine the tissue specificity or tropism of a viral vector. Accordingly, the capsid serotypes for use in the invention will typically be one that has natural tropism for or a high efficiency of infection of the target cells. For example, AAV8 capsid serotypes have a natural tropism for cells of the retina, whilst AAV2 and AAV9 have a natural tropism for neurons. The vector may comprise an AAV8 capsid coat or a derivative thereof.

Chimeric capsid proteins include those generated by recombination between two or more capsid coding sequences of naturally occurring serotypes. This may be performed for example by a marker rescue approach in which non-infectious capsid sequences of one serotype are cotransfected with capsid sequences of a different serotype, and directed selection is used to select for capsid sequences having desired properties. The capsid sequences of the different serotypes can be altered by homologous recombination within the cell to produce novel chimeric capsid proteins.

Chimeric capsid proteins also include those generated by engineering of capsid protein sequences to transfer specific capsid protein domains, surface loops or specific amino acid residues between two or more capsid proteins, for example between two or more capsid proteins of different serotypes.

Shuffled or chimeric capsid proteins may also be generated by DNA shuffling or by error-prone PCR. For example, hybrid AAV capsid genes can be created by randomly fragmenting the sequences of related AAV genes e.g. those encoding capsid proteins of multiple different serotypes and then subsequently reassembling the fragments in a selfpriming polymerase reaction, which may also cause crossovers in regions of sequence homology. A library of hybrid genes created in this way by shuffling the capsid genes of several serotypes can be screened to identify viral clones having a desired functionality. Similarly, error prone PCR may be used to randomly mutate capsid genes to create a diverse library of variants which may then be selected for a desired property.

The sequences of the capsid genes may also be genetically modified to introduce specific deletions, substitutions or insertions with respect to the native wild-type sequence. In particular, capsid genes may be modified by the insertion of a sequence of an unrelated protein or peptide within an open reading frame of a capsid coding sequence, or at the N- and/or C-terminus of a capsid coding sequence.

The unrelated protein or peptide may advantageously be one which acts as a ligand for a particular cell type. It may thereby confer improved binding to a target cell or improve targeting or the specificity of targeting of the vector to a particular target cell population, for example, photoreceptor cells of the retina. In other cases, the unrelated protein may be one which assists purification of the viral particle as part of the production process i.e. an epitope or affinity tag. The site of insertion will typically be selected so as not to interfere with other functions of the viral particle e.g. internalisation, trafficking of the viral particle. The skilled person can identify suitable sites for insertion based on their common general knowledge. Particular sites are disclosed in Choi et al., referenced above. The vectors or particles also includes chemically modified forms bearing ligands adsorbed to the capsid surface. For example, such ligands may include antibodies for targeting a particular cell surface receptor.

In some cases, the viral or non-viral vectors described herein may be packaged in a vesicle, liposome, exosome or nanoparticle or other suitable means of packaging as are known to those skilled in the art.

Nucl ease-deactivated Cas13 The vectors of the invention encode a dCas13-ADAR _DD fusion protein comprising nucl ease-deactivated Cas13 (dCas13) and the deaminase domain of an adenosine deaminase acting on RNA (ADAR _DD ).

The type VI CRISPR nuclease Cas13 binds to single stranded RNA directed by a programmable CRISPR RNA (crRNA) guide. In their active form, the Cas13 enzyme family act as ribonucleases (RNase) following recognition of a target RNA sequence, cleaving target RNA transcripts. RNase activity is mediated by two higher eukaryotes and prokaryotes nucleotide-binding (HEPN) RNase domains. Mutation to key conserved catalytic residues in the two HEPN domains deactivates the RNase activity of Cas13, resulting in a catalytically deactivated protein capable of programmable RNA binding (dCas13). Hence, the dCas13 used in the invention has one or more mutations in a HEPN domain resulting in deactivation of the RNase activity.

A Cas13b orthologue derived from Prevotella sp. P5-125 (PspCas13b) has been fused to ADAR2DD to create a programmable RNA editor termed REPAIR (RNA Editing for Programmable A to I Replacement) (Cox et al. 2017 Science). A guide RNA is used to direct dCas 13b binding to the sequence of interest (Figure 1 ), creating a targeted doublestranded RNA substrate for the fused ADAR _DD . Without wishing to be bound by theory, it is believed that the direct repeat hairpin of the gRNA recruits the Cas13-ADAR _DD fusion to the target site for A>I editing, as described below. Hence in some cases in accordance with the invention, the dCas13 is a dCas13b such as PspCas13b, or a truncated version thereof, as described below, and/or the ADAR _DD is an ADAR2DD.

The full-length REPAIR fusion (dPspCas13b-ADAR2 _DD ) is 4.47kb and unable to fit in the AAV coding capacity together with the required regulatory elements. Hence, according to the present invention, a truncated version of dCas13 may be used. In some cases the truncated version is C-terminal truncated. In some cases the dCas13 has a deletion of residues 984 to 1090. In some cases, the C-terminal truncated dCas13 may have the sequence of SEQ ID NO: 21 (dCas13bA984-1090), or be a variant having at least 80%, or at least 85%, 88%, 90%, 92%, 95%, 98%, or 99% sequence identity therewith. The sequence encoding the dCas13 may for example have the sequence of SEQ ID NO: 3.

In other cases, the dCas13 is an orthologue of Cas13bt, such as those described in Kannan et al. (Nat Biotechnol. 2021). In some cases, the dCas13b orthologue has the amino acid sequence encoded by any one of Cas13btl (SEQ ID NO: 4), Cas13bt3 (SEQ ID NO: 5), and Cas13bt5 (SEQ ID NO: 6), or is a variant thereof having at least 80%, or at least 85%, 88%, 90%, 92%, 95%, 98%, or 99% sequence identity therewith. In a specific example, the vector comprises a nucleotide sequence that encodes the Cas13bt3 -ADAR _DD fusion protein operationally linked to a rhodopsin kinase promoter and/or a SV40 polyadenylation signal. The vector may further comprises a nucleotide sequence that encodes the guide RNA operationally linked to a U6 promoter.

The variant of dCas13 used for the invention is one that retains the ability to create a double stranded target RNA with the guide RNA for the enzymatic activity of the fused ADAR _DD .

The dCas13 may comprise one or more Nuclear Localisation Signals (NLSs) or Nuclear Export Signals (NES), typically at the N-terminal and/or the C-terminal.

Adenosine deaminase acting on RNA (ADAR) enzymes

ADARs act to convert adenosine residues to inosines (A>I editing) in doublestranded RNA. Inosine is structurally similar to guanosine and is read as a guanosine by most cellular machinery including during translation, splicing and reverse transcription, effectively creating an A>G edit in RNA. All ADARs have two common structural motifs. A double-stranded RNA-binding domain (dsRBD) makes direct contact with double stranded RNA (dsRNA), while a C-terminal deaminase domain (ADAR _DD ) catalyses hydrolytic deamination where an amino group is replaced by a hydroxyl group, converting adenosine to inosine. This allows inosine to act as guanosine and pair with cytidine by normal base-pairing. A base-flipping mechanism rotates the target adenine base from the RNA helix into the enzyme catalytic pocket to allow the enzyme to attack the target C6 carbon.

In the vectors of the invention, the dsRBD is replaced by the amino acid sequence of a nucl ease-deactivated Cas13, as described above, to produce the dCas13b-ADAR _DD fusion protein, typically with a linker between the dCas13b and the ADAR _DD as described below. Any suitable ADAR _DD may be used. Two ADAR enzymes have been identified in humans to carry out A>I editing activity, AD ARI and ADAR2. AD ARI is expressed as two isoforms, a shorter 110kDa isoform (AD ARI p110) and a larger 150 kDa isoform (ADAR1 p150). AD ARI p110 is constitutively expressed and localizes to the nucleus, while AD ARI p150 is inducible by activation of the innate immune sensing system and localizes to the cytoplasm. AD ARI is expressed ubiquitously including in the retina. ADAR2 is expressed predominantly as a single isoform and nuclear localization signals mediate the localization of ADAR2 primarily to the nucleus and nucleolus. ADAR2 is most strongly expressed in the lung and brain. Evidence suggests that ADAR2 RNA is expressed in the retina and immunohistochemistry demonstrates ADAR2 localised to retinal ganglion cells.

In some cases the ADAR _DD of the fusion protein used in the invention may have the amino acid sequence of the human ADAR2DD (SEQ ID NO: 10) or be a variant thereof The variant may, for example, have at least 80%, or at least 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 10. The variant retains enzymatic activity. In particular cases, the variant includes a substitution of residue E488 of hADAR2 (residue 173 of SEQ ID NO: 10), such as E488Q, or the ADAR _DD may have the sequence of SEQ ID NO: 22. Hence the vector may, for example, comprise the sequence of SEQ ID NO: 8. The E488 residue of ADAR2 stabilizes the base-flipped structure by taking the space of flipped adenosine and hydrogen bonding to the opposite base. The E488Q hyperactive mutant displays increased editing activity, likely because glutamic acid (Q) is relatively more protonated than glutamine (E) and thus makes it a better hydrogen bond donor to bind an opposing cytidine base. In the ADARIDD, the E488Q mutation has been identified as a hyperactive mutation with similar activity to the E488Q mutation in ADAR2DD. Further engineering has produced variants with improved specificity. The T375 residue helps stabilize the edited strand in the cleft containing the active site through hydrogen bonding to the 2’-hydroxyl of the flipped base and the phosphate between the flipped base and the +1 base. An engineered E488Q/T375G ADAR2DD mutant displays much greater specificity and relatively preserved efficiency for editing of the target adenine. Hence, in some cases the ADAR _DD variant used in the invention includes a substitution of both residue E488 and T375 of hADAR2, for example E488Q and T375G, or the ADAR _DD may have the sequence of SEQ ID NO: 23. Hence the vector may, for example, comprise the sequence of SEQ ID NO: 9.

In some cases the dCas13-ADAR2 _DD fusion protein may comprise or consist of the sequence of SEQ ID NO: 14 (dPstCas13b-AADAR _DD (E488Q) with FLAG tag), SEQ ID NO: 38 (FLAG tag typically removed for clinical use) or SEQ ID NO: 39 (dPstCas13b- AADAR _DD (E488Q, T375G), or be a variant having at least 90%, or at least 95%, 98% or 99% sequence identity thereto.

Linkers

A linker sequence is typically included in between the sequence encoding the dCas13 and the sequence encoding the ADAR _DD . The linker may be any flexible region/moiety that allows the fusion protein to flex and hence generally improves binding. A suitable linker sequence may, for example comprise or consist of one or more glycines followed by a serine, and repeats thereof (for example up to 3 or 4 repeats), for example GS, GGS, GGGS, (GGS)2 and (GGS)3. In a particular embodiment, the linker in a vector expressing both the fusion protein and the gRNA may be (GGS)2. In a vector encoding just the fusion protein and not the gRNA the linker may be GS, or the vector may comprise two GS linkers flanking a NES. Other suitable linker sequences are described in Cox et al. (2017).

Nuclear Export Signal

One or more (for example, two or three) nuclear export signal sequences (NES) is typically included in the vector. For example, a NES maybe included at the 5’ or 3’ end of the sequence encoding the dCas13 or adjacent to one or more linker sequences as described herein. An example is the HIV NES (reference sequence SEQ ID NO: 34). Other examples are the MARK NES (SEQ ID NO: 35), the PKI-alpha NES (SEQ ID NO: 36) or the FAK NES2 (SEQ ID NO: 37) .

Guide RNA

The guide RNA (also referred to as a CRISPR RNA or crRNA, or alternatively as an ADAR guiding RNA or adRNA) used for the invention may be encoded either on the same vector as the dCas13-ADAR _DD fusion protein, or on a separate second vector. The guide RNA binds to a target RNA expressed in the retina. The target RNA is a variant that is pathogenic (comprises a pathogenic mutation), usually because it encodes or produces a defective protein or fails to encode an active protein that is present in healthy cells/retina. Most typically the RNA is encoded by a recessive gene, or a gene that contributes to recessive disease in the retina. The gene may be a large gene, for example larger than about 4.2 kb, which is generally not amenable to AAV-mediated gene replacement. Examples include the genes ABCA4, USH2A, CEP290, MY07A, EYS and CDH23. Most typically the gene is USH2A, or human USH2A. Hence, the target RNA is USH2A or hUSH2A.

The guide RNA comprises and typically consists of a 3’ end hairpin or stem loop structure and a 5’ end tail or spacer region. The hairpin/stem loop structure is formed by a pair of palindromic/direct repeat sequences, typically about 36 nucleotides in length (e.g. 35-37 nucleotides), but may be 20-50 nucleotides in length, or 25 to 45 or 30 to 40 nucleotides in length. Typically the stem sequence has GTTG/GUUG at the 5’ end reverse complementary to a CAAC at the 3’ end. The sequence forming the loop or bulge between the palindromic sequences is typically about 3 to 10, or more typically 4 to 8, or about 6 nucleotides in length. The spacer region, also known as the homology region, is designed to be capable of hybridizing to a target section of the target RNA.

The spacer is typically 30 to 84 nucleotides in length. That is, the guide RNA binds to the target RNA over a length of 30 to 84 nucleotides, or more typically 30 to 60 nucleotides, or 40 to 60 nucleotides, or 45 to 55 nucleotides, or 49 to 51 nucleotides, or about 50 nucleotides. The spacer has a sequence that is generally reverse complementary to that of the target region of the target RNA to which it binds/hybridizes. However, the spacer sequence includes a (i.e. at least one) mismatch between the guide RNA and the target RNA sequence (nucleotide sequence mismatch, e.g. a single nucleotide sequence mismatch). The spacer sequence generally includes a cytidine nucleotide mismatched to an adenosine in the target RNA. This is the adenosine that is the target of A>I conversion by the ADAR _DD . Other mismatches may be included as long as the guide RNA still hybridizes to the target sequence. For example, additional mismatches could be introduced to reduce off-target edits. Hence, in some cases, mismatches may make up to 2%, 4%, 6%, 8% or 10% of the spacer sequence, or there may be up to 1, 2, 3, 4, 5 or 6 mismatches in addition to the mismatch that defines the target site for the A>I conversion. However, in some cases, there are at least 2, or at least 3, 4 or 5 matched (reverse complementary) nucleotides in the guide RNA either side of the mismatch.

The mismatched nucleotide that defines the target site for the A>I conversion mismatch distance is typically at least 4, or at least 5, 6, 7, 8, 9 or 10 nucleotides from the 3’ and/or 5’ end of the spacer sequence. The “mismatch distance” is the number of nucleotides between the stem sequence and the mismatch nucleotide in the spacer sequence that defines the target site for the A>I conversion (Figure 2C). The mismatch distance is typically between 18 and 42 nucleotides, or more typically between 30 and 40 nucleotides, or 34 and 38 nucleotides, or 35 and 37 nucleotides, or is about 36 nucleotides. In a particular embodiment, the spacer sequence is about 50 nucleotides in length (or between 45 to 55 nucleotides) and the mismatch distance is about 36 nucleotides (or between 35 and 37 nucleotides). In another embodiment, the spacer sequence is about 50 nucleotides in length (or between 45 to 55 nucleotides) and the mismatch distance is about 38 (or between 37 to 39 nucleotides).

The nucleotide sequence mismatch between the guide RNA and the target RNA that defines the target for A>I conversion is at a site corresponding to a pathogenic mutation or variant. For example the site might correspond to a G>A variant in NCBI’s ClinVar Database (https://www.ncbi.nlm.nih.gov/clinvar/) (and classed as “pathogenic”, or classed as “pathogenic” or “likely pathogenic”). In the most typical cases, the pathogenic mutation produces a premature stop codon in the target RNA, or a premature stop codon in the first 95%, 90%, 85%, 80%, 75%, 70%, 65% or 60% of the coding sequence. For example a G>A mutations in tryptophan codons that produce premature stop codons such as TGG>TGA/TAG/TAA (p.Trp>Ter). In other cases, the (G>A) mutation my cause a missense mutation.

In some embodiments, the site corresponding to a pathogenic mutation or variant is an adenine at a site homologous to c.l 1864G in SEQ ID NO: 42 (reference Homo sapiens usherin (USH2A), mRNA transcript variant 2 CDS; https://www.ncbi.nlm. nih.gov/gene?Db=gene&Cmd=DetailsSearch&Term=7399). c,12304G in SEQ ID NO: 1 (RefSeq Transcript NM 206933.4; https://www.ncbi.nlm.nih.gOv/nuccore/NM_206933.4), and position 153 in SEQ ID NO: 2 (ref|NM_206933.4|:12151-12505 Homo sapiens usherin (USH2A), transcript variant 2, mRNA Exon 61). The transcript NM 206933.4 contains an untranslated region prior to the start codon. The start codon (where the CDS begins) is at position 440 in the RefSeq transcript. So the equivalent position of c.l 1864 in the cDNA is position 12304 in the RefSeq Transcript. The spacer sequence of the guide RNA binds to (i.e. is capable of hybridizing to) a region of exon 61 of the human USH2A sequence (SEQ ID NO: 2) that includes the c.11864A variant. Typically the target RNA sequence bound by the guide RNA includes at least 3, or at least 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides either side of the adenine. Typically, the spacer sequence includes a cytidine at the site corresponding to c.l 1864G (position 153 in SEQ ID NO: 2) and reverse complementary sequence to that in SEQ ID NO: 2 for at least 3, or at least 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides either side (3’ and/or 5’) of the cytidine. Hence, the spacer sequence typically includes the sequence GACCACA, or the sequence TGACCACAG, ATGACCACAGA, AATGACCACAGAC, TAATGACCACAGACT, TTAATGACCACAGACTC, GTTAATGACCACAGACTCT, or GTTAATGACCACAGACTCT. In a specific embodiment the guide RNA spacer comprises or consists of the sequence SEQ ID NO: 20, or a sequence having at least 80%, or at least 85%, 90%, 95%, 98% or 99% sequence identity therewith, and typically one or more of the sequences set out above. In some cases the guide RNA has the sequence of SEQ ID NO: 32, or a sequence having at least 70%, or at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity therewith. Other features of the guide RNA may typically be as described above.

Promoters and other regulatory elements

In the vector, the nucleic acid encoding the fusion protein is typically operably linked to a promoter. The term “operably linked” means that the regulatory element is present at an appropriate position relative to another nucleic acid sequence (such as a transgene) so as to effect expression of that nucleic acid sequence., i.e. in their intended manner. A control sequence (e.g. a promoter) “operably linked” to a transgene is ligated in such a way that expression of the transgene is achieved under conditions compatible with the control sequences.

In some cases the promoter may be constitutive i.e. operational in any host cell background, for example, the ubiquitous CAG promoter. More typically, the promoter is a cell-specific promoter, which drives expression in a particular target cell type, for example photoreceptor cells of the retina. Examples of suitable promoters include the human rhodopsin kinase promoter (GRK1), which may have the sequence of SEQ ID NO: 11, or functional variants thereof. Other promoters that are described herein and that may be used where appropriate (i.e. where space in the vector allows) are the minimal elongation factor-1α (EPS), cytomegalovirus (CMV-IE) and the minimal synthetic super core promoter 1 (SCP1) and the human rhodopsin promoter (which may have the sequence of SEQ ID NO: 28).

The nucleic acid encoding the fusion protein is typically operably linked to a polyadenylation signal. Examples of polyadenylation signal sequences that could be used include the highly minimal soluble neuropilin- 1 (sNRPl, 32 bp) polyadenylation signal (reference sequence SEQ ID NO: 13) such as described in McFarland et al. Plasmid. 2006;56:62-7, bovine growth hormone (bGH, 225 bp) polyadenylation signal (reference sequence SEQ ID NO: 31) and the SV40 polyadenylation signal sequence of (reference sequence SEQ ID NO: 18).

The nucleic acid encoding the gRNA is typically operably linked to a promoter, typically a type III RNA polymerase III promoter, such as U6 (hU6, reference sequence SEQ ID NO: 12). Another promoter that may be used where appropriate and that is described herein is the strong constitutive tRNAcLN (tRNAscan-SE-ID: chr15.tma7, 70 bp) promoter, such as described in Knapp et al. Nat Commun. 2019; 10. One or more other regulatory elements, such as enhancers, post-regulatory elements and polyadenylation sites may also be present in addition to the promoter. A regulatory sequence that is operably linked to the transgene is any sequences that facilitates or controls expression of the transgene, for example by promoting or otherwise regulating transcription, processing, nuclear export of mRNA or stability.

A vector of the invention may typically comprise the following elements (in a 5’ to 3’ direction unless otherwise indicated): (a) an inverted terminal repeat sequence (5’ITR), such as any ITR sequence or 5’ITR sequence described herein, or the sequence of SEQ ID NO: 26, or a variant having at least 70%, or 80% or 85% or 90% or 95 % or 98% or 99% sequence identity to SEQ ID NO: 26; (b) a promoter sequence, wherein the promoter is operably linked to a sequence encoding the dCas13-ADAR _DD fusion protein, for example the GRK1 promoter comprising the sequence of SEQ ID NO: 11; (c) a translation initiation sequence, such as the Kozak consensus sequence GCCACC; (d) optionally a chicken betaactin promoter exon-intron-exon sequence (Ex/In/Ex), such as the sequence of SEQ ID NO: 29, or a variant having at least 70%, or 80% or 85% or 90% or 95 % or 98% or 99% sequence identity to SEQ ID NO: 29, wherein the Ex/In/Ex sequence is 3’ or 5’ to the translation initiation sequence; (e) a sequence encoding the dCas13-ADAR _DD fusion protein, such as the sequence of SEQ ID NO: 14 (with FLAG tag) or 38 (FLAG tag typically removed for clinical use); (f) optionally a nuclear export signal (NES), such as the HIV NES having the sequence of SEQ ID NO: 15; (g) optionally a woodchuck hepatitis post-transcriptional regulatory element (WPRE) having the sequence of SEQ ID NO: 30, or a variant having at least 70%, or 80% or 85% or 90% or 95 % or 98% or 99% sequence identity to SEQ ID NO: 30; (h) a polyadenylation tail sequence, such as a dual soluble neuropilin- 1 (sNRPl) polyadenylation signal sequence of SEQ ID NO: 13; and (i) a 3’ inverted terminal repeat sequence (3 ’ITR), such as any ITR sequence or 5’ITR sequence described herein, or the sequence of SEQ ID NO: 27, or a variant having at least 70%, or 80% or 85% or 90% or 95 % or 98% or 99% sequence identity to SEQ ID NO:27. These components (a) to (h) ((d), (f) and (g) being optional, i.e. each independently either present or absent) may be referred to as an expression cassette. The NES sequence (f) may also be included at the 5’ end of the sequence encoding the fusion protein instead of at the 3’ end, or in the linker of the sequence encoding the fusion protein as it is in SEQ ID NO: 38.

A further vector of the invention may typically comprise the following expression cassette/elements (in a 5’ to 3’ direction unless otherwise indicated): (a) an inverted terminal repeat sequence (5’ITR), such as any ITR sequence or 5’ITR sequence described herein, or the sequence of SEQ ID NO: 26, or a variant having at least 70%, or 80% or 85% or 90% or 95 % or 98% or 99% sequence identity to SEQ ID NO:26; (b) a promoter sequence, wherein the promoter is operably linked to a sequence encoding the gRNA, for example the U6 promoter comprising the sequence of SEQ ID NO: 12; (c) a sequence encoding the guide RNA, such as the sequence of SEQ ID NO: 32 or any other suitable gRNA sequence described herein; and (d) a 3’ inverted terminal repeat sequence (3 ’ITR), such as any ITR sequence or 5’ITR sequence described herein, or the sequence of SEQ ID NO: 27, or a variant having at least 70%, or 80% or 85% or 90% or 95 % or 98% or 99% sequence identity to SEQ ID NO:27.

In some embodiments, the invention provides both of the two vectors above provided together, for example in a kit, or for the use of both vectors in combination, as described further herein.

A further vector of the invention may typically comprise the following elements (in a 5’ to 3’ direction unless otherwise indicated): (a) an inverted terminal repeat sequence (5’ITR), such as any ITR sequence or 5’ITR sequence described herein, or the sequence of SEQ ID NO: 26, or a variant having at least 70%, or 80% or 85% or 90% or 95 % or 98% or 99% sequence identity to SEQ ID NO:26; (b) a promoter sequence, wherein the promoter is operably linked to a sequence encoding the dCas13-ADAR _DD fusion protein, for example the GRK1 promoter comprising the sequence of SEQ ID NO: 11; (c) a translation initiation sequence, such as the Kozak consensus sequence GCCACC; (d) a sequence encoding the dCas13-ADAR _DD fusion protein, such as the sequence of SEQ ID NO: 38 (e) optionally a nuclear export signal (NES), such as the HIV NES having the sequence of SEQ ID NO: 15; (f) a polyadenylation tail sequence, such as a SV40 polyadenylation signal sequence of SEQ ID NO: 18; (g) a promoter sequence, wherein the promoter is operably linked to a sequence encoding the gRNA, for example the U6 promoter comprising the sequence of SEQ ID NO: 12; (h) a sequence encoding the guide RNA, such as the sequence of SEQ ID NO: 32 or any other suitable gRNA sequence described herein; and (i) a 3’ inverted terminal repeat sequence (3 ’ITR), such as any ITR sequence or 5’ITR sequence described herein, or the sequence of SEQ ID NO: 27, or a variant having at least 70%, or 80% or 85% or 90% or 95 % or 98% or 99% sequence identity to SEQ ID NO:27. The NES sequence (f) may also be included at the 5’ end of the sequence encoding the fusion protein instead of at the 3’ end, or in the linker of the sequence encoding the fusion protein as it is in SEQ ID NO: 38. There may be intervening sequences between the some or all of the different components (a) to (x). An intervening sequence between any two adjacent elements in the sequence may in some cases be up to 200 nucleotides, or up to 150, 100, 75, 50, 40, 30, 20, 15, 10, or 5 nucleotides in length. The vector may also include additional nucleotide sequences encoding additional or alternative regulatory elements such as one or more (further) promoters or enhancers or locus control regions (LCRs). The vector may also comprise other sequence elements or remnants of sequence elements used for the construction, cloning, selection and so on of the vector, as are well known to those skilled in the art. The skilled person is able to select a suitable combination of elements to not exceed the maximum capacity of the AAV vectors for either a two vector or single vector system as described herein.

In some cases the vector comprises the sequence of any one of SEQ ID NOs: 16, 33, 24, 25 or 40.

Preparation of vector

A vector of the invention may be prepared by standard means known in the art for provision of vectors for gene/RNA therapy. Thus, well established public domain transfection, packaging and purification methods can be used to prepare a suitable vector. This includes known methods for packaging vectors into vesicles, liposomes, exosomes or nanoparticles or the like.

Viral vectors used in gene therapy are typically generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, as described above, other viral sequences being deleted, leaving capacity for an expression cassette for one or more transgenes. The missing viral functions are typically supplied in trans by the packaging cell line.

Packaging cells are typically used to form virus particles that are capable of infecting a host cell. The packaging cells may be any suitable cell type known in the art. The packaging cells are typically human or human derived cells. Suitable cells include Human Embryonic Kidney (HEK) 293 or 293T cells, or HEK 293 derived cell clones (for example to package adenovirus derived vectors), HeLa cells (for example to package HIV or other lentivirus derived vectors) and ψ2 cells or PAS 17 cells (for example to package retrovirus derived vectors). Other examples are BHK or CHO cells. AAV derived vectors of the invention may comprise the full genome of a naturally occurring AAV virus in addition to the elements for gene therapy. However, commonly a derivatised genome will be used, for instance a derivative which has at least one inverted terminal repeat sequence (ITR), but which may lack any AAV genes such as rep or cap.

In order to provide for assembly of a derivatised or recombinant genome into the viral particle, additional genetic constructs providing AAV and/or helper virus functions will be provided in a host cell in combination with the derivatised/recombinant genome. For AVV vectors, these additional constructs will typically contain genes encoding structural AAV capsid proteins i.e. cap, VP1, VP2, VP3, and genes encoding other functions required for the AAV life cycle, such as rep. The selection of structural capsid proteins provided on the additional construct will determine the serotype of the packaged viral vector. For replication incompetent viral vectors, helper virus functions, for example adenovirus helper functions, will typically also be provided on one or more additional constructs to allow for replication. The additional constructs may be provided as plasmids or other episomal elements in the host cell, or alternatively one or more constructs may be integrated into the genome of the host cell. Suitable genes and constructs may in some cases be any of those described herein.

The invention provides a host cell that produces the gene therapy vector as described herein. The host cell may have any suitable properties as described above.

The invention also provides a method for production of a vector of the invention. The method comprises providing a host cell according to the invention as described above and culturing the host cell under conditions suitable for the production of the vector. The method may comprise providing means and/or conditions for the replication of the vector and/or assembly of the vector into a viral particle and/or into other suitable packaging such as a vesicle, liposome, exosome or nanoparticle. Optionally, the method further comprises a step of purifying the vectors or viral particles and/or formulating the vectors or viral particles for therapeutic use.

The properties of the vectors and other products of the invention as described herein can be tested using techniques known by the person skilled in the art. In particular, a vector or other construct of the invention can be delivered to a test animal, such as a mouse, and the effects observed and compared to a control. Such use is also an aspect of the invention.

Methods of therapy and medical uses The vectors described herein may be used as methods of treatment. Specifically, the vectors may be used as treatment for individuals with retinal degeneration attributed to a G>A variant/ mutation in an RNA expressed in the retina, or with Usher syndrome attributed to a G>A variant/mutation in USH2A, or an A at a position homologous to c.11864G in SEQ ID NO: 1. No current treatment exists for such patients, with inevitable blindness. Such methods of treatment form part of the present invention, as do the vectors or other products as described herein for use in such treatment, and the use of the vectors or other products of the invention as described herein in the manufacture of a medicament for use in the treatments described herein. Specifically, the vectors may be used in the treatment, prevention or reversal of retinal degeneration or retinal dystrophy, or any condition associated with a loss of function of a protein expressed in the retina a subject or patient due to a G>A variant/mutation. Conditions that may be treated include retinal degeneration or retinal dystrophy, cone-rod dystrophy, cone-dystrophy, rod-dystrophy, rod-cone dystrophy (retinitis pigmentosa), macular dystrophy, or late-onset macular dystrophy, macular degeneration, central areolar choroidal degeneration or geographic atrophy or any other retinal phenotype pathology attributed to G>A sequence variants/mutations.

In some cases, the treatment achieves or is intended to achieve any one or more of the following effects:

Improvement from baseline in microperimetry o Proportion of patients with improved microperimetry

Improvements in best corrected visual acuity (BCVA) o ETDRS visual acuity chart

Improvements on retinal function on microperimetry o Change in sensitivity (dB)

Spectral domain optical coherence tomography (SD-OCT) o Changes in reflectivity of the ellipsoid zone o Changes in outer segment length (i.e. regeneration of photoreceptor outer segments) o Changes in outer nuclear layer thickness at pre-defined retinal loci (i.e. prevention of photoreceptor cell death) o Changes in the external limiting membrane

Fundus autofluorescence (AF) o To assess changes in the retina from baseline in autofluorescence imaging (i.e. reduction in the rate of central macular degeneration)

Visual Fields o Octopus 900 pro will be used to assess changes in central peripheral vison from baseline

El ectroreti nography :

Changes in rod or cone responses

Changes in macular responses (i.e. pattern ERG)

Also, where applicable for patients: regeneration of the photoreceptor outer segment; prevention of photoreceptor (rod and/or cone) cell death; reduced rate of photoreceptor (rod and/or cone) cell death; increased photoreceptor layer thickness; increased retina thickness; increased superior retina thickness; increased inferior retina thickness; increased outer retinal thickness, increased inner retina thickness; increased distance between the outer plexiform layer and the retinal pigment layer; lengthening of the photoreceptor outer segment band; thickening of the ellipsoid band; increased distance between the inner and outer boundaries of the photoreceptors; thickening or regeneration of the external limiting membrane; restoration of the photoreceptor outer segment band; improved cone and/or rod photoreceptor function; improved/increased electroretinography (A-wave amplitudes and/or B-wave amplitudes) responses; improved eyesight or vision, improved eyesight or vision at low light intensity; improved night vision; a prevention of decline in any one or more of these measurements or the prevention of blindness. Any suitable method(s) may be used to measure these outcomes.

The method of treatment may be regarded as a method of gene therapy. The term “gene therapy” means the therapeutic delivery of nucleic acid polymers into a subject, and usually to specific target cells, as discussed further below.

The subject may be a human or a non-human animal. Non-human animals include, but are not limited to, mammals, rodents (including mice and rats), and other common laboratory, domestic and agricultural animals, including rabbits, dogs, cats, horses, guinea pigs, cows, sheep, goats, pigs, chickens, amphibians, reptiles etc.

Pharmaceutical Compositions and Modes of Administration

The one or more vectors or other therapeutic products of the invention as described herein may be formulated into pharmaceutical compositions. Such pharmaceutical compositions and their use in methods of treatment as described herein form part of the invention. Pharmaceutical compositions may comprise, in addition to the vector etc., a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the vectors. Examples of suitable compositions and methods of administration are provided in Esseku and Adeyeye (2011) and Van den Mooter G. (2006). The precise nature of the carrier or other material may be determined by the skilled person according to the route of administration. Examples of techniques and protocols can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

The vectors of the invention may be administered by any suitable route and means that allows for transduction of the target cells. The target cells are rod and cone photoreceptor cells within the retina. Typically, delivery is by subretinal injection, or less commonly, by intravitreal injection or suprachoroidal injection. The vector may be delivered surgically beneath the retina, for example by sub-retinal injection.

For injection at the site of affliction, the active ingredient will be in the form of an aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as phosphate-buffered saline, Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection, Hartmann's solution. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Dosages and dosage regimes can be determined within the normal skill of the medical practitioner responsible for administration of the composition. The dose of a vector of the invention may be determined according to various parameters, especially according to the age, weight and condition of the patient to be treated; the route of administration; and the required regimen.

Administration is typically in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. For example, a therapeutically effective amount of a vector of the invention or an effective method of treatment in accordance with invention may be one that results in expression of the transgene in target cells/photoreceptor cells. Outcome measures are as described elsewhere herein above.

In other words, the treatment is sufficient to result in a clinical response or to show clinical benefit to the individual, for example to cure disease, prevent or delay onset or progression of the disease or condition or one or more symptoms, to ameliorate or alleviate one or more symptoms, to induce or prolong remission, or to delay relapse or recurrence. In some cases the treatment is sufficient to improve the subject’s eyesight. In some cases the treatment is sufficient to slow down, reduce or prevent (further) degeneration of the subject’s sight over time. The treatment may in some cases improve or reduce loss of vision in low light conditions.

A typical single dose of the one or more vectors of the invention may between 10 ⁸ , or 10 ⁹ or 2.5x 10 ⁹ or 5x10 ⁹ ; and 10 ¹⁵ , or 10 ¹⁴ , or 10 ¹³ , or 10 ¹² , 10 ¹¹ or 5x10 ¹⁰ or 2.5x10 ¹⁰ or 10 ¹⁰ viral genomes (vg), or any range thereof, such as 2.5x10 ⁹ to 5x10 ¹⁰ vg. A dose at the lower end of these ranges will typically be used for administration direct to the retina, whilst a dose at the higher end of the range will typically be needed for systemic administration.

The dosing range of vectors used for retinal gene therapy in patients is determined through phases 1-3 of clinical trial.

A single AAV capsid that contains a single stranded DNA molecule is a single viral genome (vg). Vg can be quantified by any suitable method as well known in the art, for example real-time PCR. The one or more vectors are preferably administered only once, resulting, depending on the vector used, in permanent or transient knock down of the target gene, but repeat administrations, for example in future years and/or with different serotypes may be considered.

A composition of the invention may be administered or for administration alone or in combination with other suitable therapeutic compositions or treatments. Published data exist regarding the safety of adjunctive substances, such as blue dye to aid subretinal delivery (PMID: 28706756), or hydroxychloroquine to augment the efficacy of gene therapy (PMID: 31309129).

Where two vectors are used, i.e. one for expressing the dCas13-ADAR _DD fusion protein and the other for expressing the gRNA, both vectors may be administered simultaneously, or in the same formulation/mixture, or separately/from different formulations/sequentially/via different routes, for example within about 6 months, 1 week, 1 day, 1 hour or 30 minutes.

Kits and Panels

The vectors, pharmaceutical compositions or other products of the invention as described herein may be provided as or packaged into a kit or provided as a panel. The pharmaceutical compositions of the kit or panel are intended/ provided for use together as one therapeutic treatment (but not necessarily for simultaneous administration, as described elsewhere herein). For example, where two vectors are used, one for expressing the dCas13-ADAR _DD fusion protein and the other for expressing the gRNA, the kit or panel may include both vectors, for example in separate containers. The kit may additionally comprise suitable means for administration and/or instructions for use, for example in an method described herein. Such kits and panels are an aspect of the present invention.

Variants

In some cases, the invention relates to variants of reference nucleotides or polypeptides described herein or the sequences thereof. Polynucleotide sequences may for example comprise one or more (for example up to 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40 or 50) amino acid additions, substitutions or deletions compared to the reference sequence. In some cases, the polypeptide sequence, for example the dCas13-ADARDD fusion protein polypeptide sequence, may also have additional sequence elements or tags at the 5’ or 3’ end or in the linker. Substitutions preferably introduce one or more conservative changes, which replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace. Alternatively, the conservative change may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and may be selected in accordance with the properties of the 20 main amino acids as defined in Table 1 below.

Table 1 - Chemical properties of amino acids

For the purpose of this invention, in order to determine the percent identity of two sequences (such as 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 amino acids 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 (z.e., % identity = number of identical positions /total number of positions in the reference sequence x 100).

Typically the sequence comparison is carried out over the length of the reference sequence. If the sequence is shorter than the reference sequence, the gaps or missing positions should be considered to be non-identical positions. Any sequence described herein as an exemplary or typical sequence in accordance with the invention may be considered a reference sequence, regardless of whether it is explicitly referred to as such.

The skilled person is aware of different computer programs that are available to determine the homology or identity between two sequences using a mathematical algorithm. In an embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (1970) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http://www.accelrys.com/products/gcg/), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Other examples of suitable programs are the BESTFIT program provided by the UWGCG Package (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, 387-395) and the PILEUP and BLAST algorithms c (for example used on its default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Variants also include truncations, wherein a part of the sequence is deleted from the 5’ or 3’ end. Any truncation may be used so long as the variant is functional as described herein. Truncations will typically be made to remove sequences that are non-essential for function in vivo and/or do not affect conformation of the folded protein, in particular folding of the active site or relevant binding site. Appropriate truncations can routinely be identified by systematic truncation of sequences of varying length from the N- or C- terminus.

Examples

Example 1 - dPspCas13b-ADAR efficiently edits USH2A premature termination codons The dPspCas13b-ADAR _DD system (Cox et al. 2017) was characterized to investigate the potential for Cas13-ADAR RNA editing systems for the repair of mutations in the RNA of retinal genes.

Firstly the editing efficiency of dPspCas13b-ADAR _DD systems for editing the human USH2A p.W3955X and the mouse Ush2a p.W3947X premature termination codon in vitro was investigated. As the expression of USH2A is confined to photoreceptor and inner hair cells and USH2A is not expressed in commonly cultured cell lines, a dual luciferase assay was developed to report A-I editing activity for screening of constructs and gRNAs in HEK293T cells (Figure 2A). From a single transcript, renilla was expressed as a normalization control, a downstream ‘target cassette’ containing the premature termination mutation of interest was cloned between flanking F2A sequences, while downstream of the target cassette, firefly was encoded as a reporter of editing activity. At baseline, the upstream termination codon prevents firefly expression, but repair of the premature stop codon to a non-stop amino acid codon allows translation to proceed and firefly is expressed. Amounts of transfected plasmid were optimised to obtain efficient editing, with 150ng of gRNA and 75ng of dPspCas13b-ADAR _DD found to be optimal in triple transfection with 50ng of luciferase plasmid (Figure 3). gRNAs to USH2A p.W3955X and Ush2a p.W3947X were screened using the dPspCas13b(A984-1090)-ADAR _DD (E488Q) (SEQ ID NO: 14) construct (henceforth dPspCas13b(del)-E488Q), containing a C-terminally truncated PspCas13b (SEQ ID NO: 3) to enable downstream AAV packaging, and ADAR _DD with the hyperactive E488Q mutation (SEQ ID NO: 8). An A-C mismatch was encoded at the target adenosine, with 50nt gRNAs tiled across the target mutation varied by distance between the mismatched base and the gRNA scaffold (mismatch distance, range 18-42nts). For the human USH2A target, a guide with a 36nt mismatch distance (50nt-36) was most active with a 38% editing rate (Figure 2D). Screening the mouse Ush2a target, broadly editing rates were poor (<20%) except for a guide with a 24nt mismatch distance (mUsh2a-24-G10) with an editing rate of 50% (Figure 2E). The limited number of 30nt guides screened showed lower editing efficiency relative to the 50nt guides.

Example 2 - Effect of dPspCas13b C -terminal truncation and ADARpp mutations.

The C -terminally truncated dPspCas13b(del) construct is relatively uncharacterized compared to the full length dPspCas13b. Using the guides identified in Example 1, the two dPspCas13b constructs were compared using both hyperactive ADARpp(E488Q) (SEQ ID NO: 8) variant and also the more specific double mutant ADARpp(E488Q/T375G) (SEQ ID NO: 9) variant identified by Cox et al (Figure 1H).

Using the hUSH2A targeting guide (50nt-36), significantly higher editing on-target editing efficiency with the E488Q compared to the E488Q/T375G ADARpp variant for both the full length (Two-way ANOVA for effect of ADAR, Tukey’s multiple comparison testing, p=0.0006) and truncated (p<0.0001) constructs as expected (Figure II).

No loss of efficiency was observed in the C-terminally truncated Cas13b constructs compared to the full-length constructs (Figure II). With the E488Q/T375G ADARpp no significant differences were observed (Two-way ANOVA for effect of ADAR, p=0.99), while surprisingly in constructs with the E488Q variant, the C-terminally truncated construct had a mean absolute editing rate 8.3% ± 2.3 higher than the full-length construct (Two-way ANOVA for effect of Cas, Tukey’s multiple comparison testing, p=0.027).

To confirm firefly restoration was as a result of A-I editing, PCR amplicons of samples were also subject to Sanger sequencing and on-target editing rates were found to highly correlate to firefly restoration rates (Figure 4), although firefly restoration underreports editing relative to sequencing with highly efficient editing rates of 77% and 86% for USH2a and Ush2a targets respectively with the dPspCas13b(del)-E488Q construct (Error! Reference source not found. 2J).

Finally, bystander deamination of local adenosines in the target were assessed across each construct (Figure 2K). In agreement with previous reports, ² the E488Q construct demonstrated significant off-targets in both the hUSH2A (2 sites) and mUsh2a targets (8 sites). Comparatively, the E488Q/T375G deaminase mutant was more specific, editing at only the target adenosine in both targets, with no off-target editing in with non-targeting guides. Off-targeting in the mUsh2a transcript outside the gRNA duplex region also occurred in corresponding positions with the non-targeting guide and no editing was detected in samples transfected with targeting guides only.

Example 3 - RNA editing with minimal dCas13bt constructs

The recently described minimal Cas13bt orthologues are shorter and thus more amenable to packaging in AAV. ³ Three of these orthologues (Cas13btl, Cas13b3, and Cas13bt5) were tested with both 30nt and 50nt spacers in a luciferase assay screen of tiled guides against the mUsh2a p.W3947X target (Figure 2F,G). The 30nt guides all demonstrated editing rates of less than 10% but editing rates of up to 21% were seen with the 50nt guides. Of note, a 17% editing rate was achieved with guide 50nt-28 with the shortest orthologue, Cas13bt3, which is ~600nt shorter than the dPspCas13(del) construct.

Example 4 - Generation of Ush2a-W3947X mouse for RNA editing

A mouse model of Usher syndrome was developed containing the Ush2a c.l 1840 G>A (p.W3947X) premature termination codon for testing of RNA editing strategies in vivo. Mice were generated on a C57BL/6J .Cdh23 ^753A>G background to correct the age- related hearing loss Cdh23 allele present in the wildtype C57BL/6J strain to eliminate confounding any auditory phenotype due to the Ush2a mutant allele. The c.11840 G>A (p.W3947X) mutation was introduced by CRISPR-Cas9 mediated homology directed repair using pronuclear injection of zygotes ¹ with SpCas9 mRNA, sgRNA and a single stranded oligonucleotide donor template. Fo offspring were mated with C57BL/6j.Cdh23 ^753A>G wildtype animals and successful transmission of a single copy of the mutant allele at the target locus to the Fi generation was confirmed by Sanger sequencing and a ddPCR copy counting assay (Figure 5 A, Figure 6).

To examine the effect of the W3947X mutation on usherin expression in the retina, immunostaining of retinal sections and western blot of retinal protein lysates was performed. Western blot with an antibody to the C -terminus of the 590kDA isoform demonstrated a protein band of expected size in wildtype retinae, with no band detected in W3947X retinae (Figure 5B). Protein lysates derived from either wildtype or W3947X eye cups did not demonstrate an usherin band, consistent with the specific photoreceptor expression of usherin. Using immunohistochemistry on retinal sections, usherin immunoreactivity was not detected at the connecting cilium at the junction between the inner and outer photoreceptor segments in Ush2a-W3947X homozygous mice, while it was localised correctly in wildtype mice (Figure 5C). These results support that mice homozygous for Ush2a c.11840 G>A (p.W3947X) demonstrate an absence of full-length usherin protein in the retina, suitable for testing of strategies to repair the underlying mutation and restore usherin expression.

Example 5 - Generation of of RNA editing transgenes for AAV delivery

Constructs were generated to enable delivery of the Cas13 transgenes using AAV. For the delivery of the larger dPspCas13-E488Q construct (4.15 kb), a dual vector approach was designed with the gRNA delivered on a separate vector together with GFP under a ubiquitous CAG promoter to identify successfully transduced cells (Figure 7 A). To fit within the ~4.8kb packaging capacity of an AAV vector, minimal promoter and polyA elements were tested, and the effect of these on editing rates was compared using the luciferase assay. Driving dPspCas13-E488Q, the minimal elongation factor-la (EFS, 212 bp) promoter maintained equivalent editing rates to the cytomegalovirus (CMV-IE, 588 bp) promoter, however rates with the minimal synthetic super core promoter 1 (SCP1, 80 bp) ^5,6 promoter were reduced by a mean of 45% (Figure 8A). Using dual copies of the highly minimal soluble neuropilin- 1 (sNRPl, 32 bp) ⁷ polyadenylation signal only resulted in a slight and non-significant reduction in editing activity compared to when the robust bovine growth hormone (bGH, 225 bp) polyadenylation signal was used (Figure 8B). Using the strong constitutive tRNA _GLN (tRNAscan-SE-ID: chr15.trna7, 70 bp) promoter ^7,8 resulted in marked reduction in editing rates (45-76% reduction) relative to the human U6 promoter (hU6, 249bp) (Figure 8C). Based on these data, both an AAV-EFS- dPspCas13b-sNRPl and an AAV-hU6-gRNA-CAG-GFP (SEQ ID NO: 41) plasmid were generated with transgene sizes within the packaging capacity of AAV (Figure 7 A). No loss of editing efficiency was observed in HEK293T cells with these minimal element constructs relative to the original plasmids using the more robust CMV-IE promoter and bGH polyA (Figure 8D). Finally, western blot and immunocytochemistry demonstrated these plasmids to produce the dPspCas13b-ADAR protein at the expected size and correctly localised to the cytoplasm (Figure 8E-F).

Having demonstrated that these AAV-EFS-dPspCas13b-sNRPl editor constructs can drive efficient RNA editing, a further construct was made, replacing the EFS promoter with the rhodopsin kinase (RK, 199 bp) promoter (Figure 7 A), used widely in retinal gene therapy clinical trials to enable photoreceptor-specific expression of the construct. Lastly, dCas13bt3 was chosen for packaging within an all-in-one plasmid as it is the smallest of the tested orthologues. This allowed space for packaging with an RK promoter, a stronger SV40 polyA terminator, and the U6-gRNA (Figure 7B) in a single vector.

Example 6 - In vivo editing of Ush2a with AAV-delivered Cas13-ADAR

Three designed Casl 3-based RNA editing AAV approaches were then used for in vivo testing in the Ush2a-W3947X mouse. The first and second approaches included the dPspCas13b-E488Q vector driven by either the EFS or RK promoter, delivered in a dual vector approach with the gRNA on a second vector. The third approach included dCas13bt3-E488Q with the gRNA encoded in an all-in-one vector. All vectors used a fused ADAR _DD (E488Q) effector. Vectors were delivered by subretinal injection to 4-week-old mice, with retinal RNA extraction performed at 4 weeks post-injection, protein extraction at 6 weeks post-injection and histology at 8 weeks post-injection (Figure 7B).

In eyes where a gRNA-GFP vector was injected, successful injection and transduction of both the targeting and non-targeting vectors was confirmed by widespread retinal GFP expression detected using en face in vivo imaging with blue autofluorescence (BAF) (Figure 7C). Expression of the transgene from each construct was further confirmed by detection of full-length dCas13-ADAR _DD transcripts with RT-PCR (Figure 7D, Figure 9) performed on RNA from injected retinas, with transcript identity confirmed by Sanger sequencing (Figure 9).

On-target editing efficiency was quantified by targeted deep sequencing with NGS (Figure 7E). The EFS-dPspCas13b vector demonstrated a mean 0.93% ± 0.57 editing rate in eyes injected with the target vector, however editing was only observed in 2 of 5 eyes. Using the RK promoter, the RK-dPspCas13b vector demonstrated a 2.04% ± 0.16 editing rate. The all-in-one RK-dCas13bt3 vector achieved a barely detectable editing rate of 0.32% ± 0.06. On-target editing was not-detectable in retinas injected with non-targeting gRNAs or those injected with PBS buffer.

Immunofluorescence analysis of retinal sections from injected eyes were used to detect usherin restoration. Eyes injected with the dual vector RK-dPspCas13-E488Q vectors, which showed the highest editing rates, demonstrated a low level of usherin restoration in the GFP positive injected area (Figure 12), observed as punctate labelling at the distal inner segment, which was not present in non-targeting or PBS injected animals (Figure 7F). Restoration of usherin was not observed in retinas that were injected with either EFS-dPspCas13b or RK-dCas13bt3 vectors (Figure 12), concordant with the low editing rates observed with these vectors.

Example 7 - Off-target editing of Ush2a with AAV-delivered Cas13-ADAR

To explore off-target conversion of bystander editing of adenosines in the Ush2a transcript, the rates of A>G conversion were analysed in vector injected eyes relative to PBS injected eyes. (Figure 10). In eyes injected with the dPspCas13b and the targeting gRNA, off-target sites editing occurred with a mean (± SEM) editing rate of 0.05% ± 0.01 for the EFS-dPspCas13b-E488Q and 0.10% ± 0.03 for RK- dPspCas13b-E488Q. Off- target editing with dPspCas13b clustered in the dsRNA region created by gRNA binding. The highest rates of edited sites in injected eyes were positions A ₃₅ and A ₃₆ (also identified in the in vitro analysis in Figure 2K as A26 and A27) which showed editing rates up to 0.36% in the RK-driven vector and are predicted to produce missense changes in the protein. A greater rate of editing and greater number of detected editing sites were detected in the RK-driven vectors than with the EFS-driven vectors, which may reflect higher expression levels of the RK vector and may also reflect a floor effect due to the low editing rates achieved. Off-target editing at sites distant to the gRNA binding site was seen across both constructs, and additionally, at two sites in eyes injected with the non-targeting RK- dPspCas13b vector.

Off-target editing rates in the Cas13bt3 -injected eyes exhibited were lower, but across a greater and more sporadic number of sites outside the gRNA binding region, observed in both the targeting and non-targeting constructs, demonstrating non-specific deamination of the transcript.

Mapping of the deep-sequencing reads enabled an analysis of the number of transcripts that contained the desired TAG > TGG edit only, without potentially deleterious off-target deamination events, an estimate of the precision of the editing (Figure 10C). This demonstrated that with RK-dPspCas13b, 71% of the transcripts with a correct A>G edit at the target locus were corrected to the wildtype sequences without any other A>G edits within the analysed region. A similar rate of 73% was seen in eyes injected with EFS- dPspCas13.

Example 8 - Effect of AAV-delivered Cas13-ADAR on Retinal Structure

Optical coherence tomography (OCT) in vivo imaging was performed at four-weeks post injection to examine for any early deleterious effects of subretinal delivery of AAV- Cas13-ADAR on outer retinal structure. Measurements of photoreceptor layer (PRL) thickness at the injected area (superior) were compared to the uninjected area (inferior) as an internal control, as well as to buffer injected animals (Figure 11).

A significant main effect of subretinal injection was observed in AAV injected eyes in the superior retina (paired two way ANOVA with retinal location and injection as factors, F(1,68) = 92.35, p < 0.0001 for effect of retinal location), with additional significant effects of injected substance (F(6,68 = 2.96, p = 0.01) and interaction between injection and location (F,(6,68) = 2.42, p = 0.04). Sub-retinal injection of a buffer control solution produced a small but non-significant 5.9% ± 4.4 (p = 0.76) loss of PRL thickness in the injected superior retina compared to the inferior retina, as expected from the surgical procedure.

The EFS-dPspCas13b-E488Q groups were injected with a 1E+9gc/eye dose of the Cas13 vector and 1E+9gc/eye of the gRNA vector. PRL thickness losses in the superior retina in the EFS-dPspCas13b-E488Q groups injected with targeting and non-targeting guides were 29% ± 6 (p = 0.0002) and 20% ± 6 (p = 0.02) respectively.

The RK-dPspCas13b groups were injected with a lower dose of the gRNA vector (1E+9gc/eye of Cas13 vector and 5E+8gc/eye of gRNA vector), however PRL thickness losses of 24% ± 5 and 26% ± 5 were observed in targeting and non-targeting gRNAs respectively. The RK-Cas13bt3 group was injected with a total dose of 1E+9gc/eye of the single vector (without GFP), and PRL losses of 12 ± 4% and 17 ± 5% were seen in the targeting and non-targeting groups respectively.

To isolate the effect of the retinal thinning, injections of the EFS-dPspCas13b-E488Q vector alone (1E+9 gc/eye), and the gRNA-GFP vector alone at 5E+8gc/eye and 1E+9gc/eye were performed. Greater retinal thinning was observed in the higher dose gRNA-GFP group than when the Cas13 vector was injected alone, or a lower gRNA-GFP vector dose was used (Figure 13).

Discussion

USH2A is the second most commonly implicated gene in inherited retinal disease, accounting for approximately 8% of IRDs (Fry et al., 2021). RNA editing offers a therapeutic strategy for correcting editable pathogenic transition variants, which account for 53% of mutations in genes such as USH2A and ABCA4, which are both common and not amenable to traditional gene replacement strategies due to their large size. The present application demonstrates the use of CRISPR-Cas13b RNA editing strategies to correct a common G>A mutation in USH2A that causes a pathogenic premature termination codon (c.l 1864G>A, p.W3955X). Furthermore, it is demonstrated that this can be used for the correction of the homologous mutation in the mouse Ush2a gene (c.l 1847G>A; pW3947X) with high efficiency. This is the first demonstration of in vivo RNA editing in the retina and the first demonstration of in vivo editing with Cas13- based RNA editors. No editing was observed when non-targeting guides or guides without DR scaffold sequences were delivered with Cas13 constructs supporting that editing was gRNA-Cas13 mediated.

Evaluation of the dPspCas13b-ADAR2DD plasmids confirmed previously published data demonstrating the robust on-target activity of the E488Q mutant ADAR and reduced on-target activity of the E488Q/T375G mutation (Cox et al., 2017). Encouragingly, the preserved editing activity seen in dPspCas13bdel constructs with the C-terminal 984-1090 deletion confirms this truncated protein retains its function for on-target editing.

Using a dual vector approach with dPspCas13b-ADAR2dd, a 2% correction of the W3947X mutation in Ush2a was achieved using a photoreceptor-specific RK promoter to drive dPspCas13b-ADAR expression. Notably, this also correlated with a restoration of usherin expression in photoreceptors in injected eyes. Although low editing rates were achieved, high levels of correction will likely not be required for the treatment of IRDs. Given the large size and stability of the usherin protein in the photoreceptor cilium, only a small amount of protein is expected to be required to improve or slow down degeneration. For instance, in two choroideremia patients, the presence of only approximately 5% of wildtype REP1 transcripts due to a non-canonical splice site mutation was associated with a markedly slower degenerative phenotype, demonstrating high levels of wildtype transcript are not necessarily required to ameliorate the disease course (Lewis E Fry et al., 2020).

Retinal thinning following injection with AAV-Cas13 constructs was also observed on in vivo OCT imaging. A proportion of the loss of photoreceptor layer thickness may be attributed to the gRNA-GFP vector. GFP is very helpful as a marker for transduction, however GFP overexpression with ubiquitous is possibly toxic to photoreceptors at doses at or exceeding le+9 gc/eye (Xiong et al., 2019). A proportion of the retinal thinning may also be due to the dCas13b and/or ADAR2DD(E488Q). Less retinal thinning was seen in the dCas13bt3 injected retinas, which may be related to the lower dose used to deliver a single rather than dual AAV vector, or may be due to the dCas13b used. In its active form PspCas13b is known to have collateral RNA cleavage and cause cell death in vivo, however this is not typically observed in the deactivated enzyme.

Sequences

SEQ ID NO : 1

RefSeq mRNA is USH2A Transcript Variant 2 NM 206933.4: https://www.ncbi.nlm.nih.eov/nuccore/NM 206933.4

SEQ ID NO: 2

>ref | NM_206933 . 4 | : 12151-12505 Homo sapiens usherin (USH2A) , transcript variant 2 , mRNA Exon 61

SEQ ID NO : 3

PspCas13b -delta 984-1090 DNA Sequence (No linker /ADAR)

SEQ ID NO: 4 Cas13btl DNA Sequence (No linker /ADAR/NES)

SEQ ID NO: 5 Cas13bt3 DNA Sequence

SEQ ID NO: 6 Cas13bt5 DNA Sequence

SEQ ID NO: 7

ADAR _DD DNA sequence reference

SEQ ID NO: 8

ADARdd E488Q DNA Sequence

SEQ ID NO: 9

ADARdd E488Q / T375G DNA Sequence

SEQ ID NO: 10

ADAR2dd WT Amino Acid Sequence

SEQ ID NO: 11

SEQ ID NO: 12 SEQ ID NO: 13

SEQ ID NO: 14

SEQ ID NO: 15

NES (HIV Rev) DNA Sequence (Nuclear Export Signal)

SEQ ID NO: 16

DNA Sequence Full RK-PspCasl3-ADAR-E488Q Vector (including AAV2 ITRs)

SEQ ID NO : 17

DNA Sequence Full U6-gRNA-CAG-GFP-WPRE cloning vector (without spacer cloned in)

SEQ ID NO: 18

SV40 PolyA DNA Sequence

SEQ ID NO: 19

AAV2/8-Y733F RepCap Gene Sequence

SEQ ID NO : 20

Guide RNA sequence - dPspCas!3b-gRNA-36

SEQ ID NO: 21

Amino acid sequence of dPspCas13 (A984-1090)

SEQ ID NO: 22

Amino acid sequence of ADAR _DD (E488Q)

SEQ ID NO: 23

Amino acid sequence of ADAR _DD (E488Q, T375G)

SEQ ID NO: 24

GRKl-dCas13-ADAR (E488Q) -sNRP vector (no ITRs, No FLAG) SEQ ID NO : 25

U6-hUsh2a-gRNA-36 complex

SEQ ID NO : 26

DNA sequence for AAV2 ITR (5' )

SEQ ID NO : 27 DNA sequence for AAV2 ITR (3' notated as reverse complement, 5' to 3' )

SEQ ID NO : 28

Human rhodopsin promoter (RHOp)

SEQ ID NO : 29

Chicken beta-actin promoter exon-intron-exon (Ex/In/Ex)

SEQ ID NO : 30 WPRE

SEQ ID NO : 31

Bovine growth hormone polyadenylation tail

SEQ ID NO : 32 gRNA hUSH2A-G3-36 (full sequence, DR, spacer)

SEQ ID NO : 33

DNA Sequence Full U6-gRNA Vector (including AAV2 ITRs) I TR-hU6 -gRNA-hUSH2 a - 36 - I TR

SEQ ID NO : 34

NES (HIV Rev) Amino Acid Sequence (Nuclear Export Signal)

SEQ ID NO : 35

MAPKK Nuclear Export Signal Amino Acid Sequence

SEQ ID NO : 36

PKI Alpha Nuclear Export Signal Amino Acid Sequence SEQ ID NO: 37

FAK Nuclear Export Signal NES2

SEQ ID NO: 38

Amino Acid Sequence dPspCas13 b-ADAR _DD (E488Q)

SEQ ID NO: 39

Amino Acid Sequence dPspCas13 b-ADAR _DD (E488Q, T375G)

SEQ ID NO : 40

RK-PspCas13b-ADARdd (E488Q-T375G) -SNRP1 - (No FLAG TAG, No ITRs)

SEQ ID NO: 41

SEQ ID NO: 42