AAV viral vectors targeting the inner ear and medical treatment of the inner ear using said vectors

Abstract

The invention provides viral vectors on the basis of adeno-associated virus (AAV) for transducing cells with a nucleic acid construct contained in the viral vector for delivery of a nucleic acid construct by the viral vector, which in their capsid proteins contain an insert arranged at an integration site for a peptide insert in viral proteins which integration site is located on a protruding loop of VP1, VP2 and / or VP3. The insert provides the viral vectors with specificity for targeting inner ear cells, especially for delivery of nucleic acid constructs contained in the viral vectors to inner ear cells.

Description

[0001] T PAATENRTANWUALTTSKTANZILESI

[0002] TARUTTIS - Aegidientorplatz 2b - D-30159 Hannover

[0003] Dr. rer. nat. Stefan Taruttis

[0004] Europaisches Patentamt Diplom-Ingenieur

[0005] Patentanwalt

[0006] 80298 MUNCHEN European Patent Attorney European Trademark Attorney

[0007] D-30159 Hannover, Aegidientorplatz 2b

[0008] TORHAUS AM AEGI

[0009] Tel.: ++49 511 123 32 670

[0010] Fax: ++49 511 123 32 678 www.taruttis.com info@taruttis.com in Kooperation mit

[0011] Dr. rer. medic Dirk Vollmer

[0012] Patentanwalt, European Patent Attorney

[0013] D-74523 Schwabisch Hall, Hilde-Domin-Str. 8

[0014] Your ReE: Ref..: Ml 111PCT 28. August 2025

[0015] New PCT Application Viral vector particle for medical treatment of the inner ear -

[0016] Medizinische Hochschule Hannover University of Kansas

[0017] Viral vector particle for medical treatment of the inner ear

[0018] The present invention relates to viral vector particles for use in medical treatment of the inner ear, especially for medical treatment of defects of the inner ear, e.g. hearing impairment, especially age-related hearing impairment, hearing impairment due to a genetic defect, and hearing impairment caused by ototoxic compounds or by exposure to intense sound. Preferably, the viral vector particles comprise a nucleic acid construct suitable for restitution of hearing, e.g. comprise a nucleic acid construct suitable for replacing or repairing a defective nucleic acid section of the inner ear and / or a nucleic acid construct suitable for expressing a protein or regulatory RNA, each of which supports restitution of hearing or maintenance of hearing or restitution or maintenance of vestibular sense. Embodiments also include use of the viral vector particle for use in the treatment of the damaged inner ear in preparation of subsequent therapeutic interventions, e.g. such as cochlear implantation. Embodiments also include use of the viral vector particles for treatment of disorders such as tinnitus and / or peripheral vestibular loss, which are also caused by dysfunction of the inner ear. Herein, reference to viral vector particles includes all embodiments of the viral vector particle, viral vector particles of one embodiment only or a mixture of at least two embodiments of the viral vector particle.

[0019] Hearing impairment which can suitably be treated by the use of the viral vector particle of the invention, which herein is also termed viral vector, can e.g. be caused by defects or impairments of supporting cells (SCs) which are present on the basilar membrane of the inner ear, defects or impairments of the inner hair cells (IHCs) or of the outer hair cells (OHCs) of the inner ear, defects or impairments of cells of the Stria vascularis of the inner ear. Genetic causes of hearing impairment include for example mutations in the GJB2 gene, the stereocilin (STRC) gene or mutations in the MY07 gene. A nucleic acid construct contained in a viral vector of the invention can comprise a gene of interest which replaces, reconstitutes or activates a defective gene of the inner ear. Examples of nucleic acids that can be delivered by the viral vector particles of the invention are nucleic acids for restoring expression of the group of wild-type genes comprising Ushlc, GJB2, MY07, Atohl, as well as the delivery of therapeutic genes including gene-editing tools such as CRIPSR-Cas system, including nucleases or nickases and single-guide RNAs with optional donor template (homology- directed repair), base editors, prime editors and RNA editors as well as growth factors found in the normal functioning inner ear, e.g. BDNF; Ntf3; GDNF; CNTF; Persephin; Neurturin and their respective receptors.

[0020] The invention provides adeno-associated viral particles which have a genetically modified capsid protein that comprises an inserted amino acid section between amino acids No. 587 and No. 588 or C-terminally directly adjacent to one of amino acids No. 453, No. 587 and No. 588 in the numbering of the wild-type capsid protein, especially of the wild-type AAV2 capsid protein (CAP, SEQ ID NO: 14), which wild-type CAP is encoded by the cap ORF (open reading frame, SEQ ID NO: 15).

[0021] The invention provides viral vectors for use in medical treatment, and medical treatment of hearing impairments or balance disturbance or balance disorder, especially treatment of impairments or defects of inner ear cells, e.g. of target cells of the group of SCs, IHCs and / or OHCs, e.g. inner sulcus cells, spiral ganglion cells, pillar cells, Deiter cells, Henssen cells, Claudius cells, and / or root cells and cells of the Stria vascularis, by administrating the viral vector of the invention comprising a nucleic acid construct suitable for replacing a defective nucleic acid section of the inner ear and / or a nucleic acid construct suitable for expressing a protein which supports restitution of hearing. Administrating the viral vector can be by injection into the vicinity or into tissue containing SCs, IHCs and / or OHCs, e.g. localized administration into the round or oval windows, by systemically administrating the viral vector particles, or preferably by localized injection into vicinity, e.g. round- or oval windows (human), or target tissue containing target cells, or injection into the superficial temporal vein. The viral vector particles can e.g. be formulated as a liquid pharmaceutically acceptable composition, e.g. in an aqueous pharmaceutical composition.

[0022] A nucleic acid construct contained in the viral vectors of the invention may comprise or consist of an expression cassette encoding a nucleic acid suitable for replacing a defective nucleic acid section of a target cell, e.g. SC, IHC and / or OHC, SGN, e.g. a nucleic acid construct suitable for genome editing, wherein a viral vector may contain a nucleic acid construct encoding an enzyme that catalyzes the editing procedure, the nucleic acid construct being combined in one viral vector or being contained in separate viral vectors, the viral vectors being combined to a mixture. Further, the nucleic acid construct may encode a regulatory RNA, may encode an antibody or may encode a growth factor. The nucleic acid construct contained in the viral vector is preferably arranged between ITRs of AAV2.

[0023] The viral vectors of the invention have been improved over wild-type AAV for inner ear cell transduction, also referred to as specificity for inner ear cells, and accordingly the viral vectors are specifically suited for targeting inner ear cells for delivering nucleic acid constructs for treatment of defects of inner ear cells or to maintain function of inner ear cells or to protect inner ear cells from damage. Therein, inner ear cells include SC, IHC, OHC and basilar membrane cells, and / or spiral SGN and / or cells of the Stria vascularis.

[0024] The viral vectors have the advantage of essentially not affecting other cells or organs but preferentially targeting inner ear cells, resulting in increase in safety and reduction of unspecific delivery by the viral vectors, e.g. to non-target cells, of nucleic acid constructs contained in the viral vector.

[0025] Background

[0026] Macdonald et al., HUMAN GENE THERAPY, Vol 32, No. 19-20, 1096-1119 (2021) describe exemplary methods for altering specific sites of the AAV2 capsid protein.

[0027] In humans, many sensorineural hearing losses are caused by genetic defects, presenting possible targets for gene therapy. Similarly, gene therapy could be used to mitigate the course of many of the diseases or could be applied as a preventive treatment option in case ototoxic drugs need to be administered to patients. Common cases of genetic deafness often result in degeneration of hair cells (HCs) and supporting cells (SCs) - most notably with GJB2 mutations (the most common hereditary deafness gene) and MY07 mutations - but degeneration of many other cell types of the inner ear is also seen, and so it is desirable to find a way to prevent the death of these cell types.

[0028] The efficacy of inner ear cell transduction using vectors consisting of the capsid proteins of naturally occurring adeno-associated viruses (AAV) is low. Therefore, the invention is of specific value.

[0029] Nicklin et al., Mol Ther 4, 174-181 (2001) describes a plasmid designated pRC99, which encodes for both, the AAV2 rep and cap ORFs, each controlled by their native promoters. This plasmid was designed to serve as helper plasmid for production of AAV vectors with modified tropism. Specifically, this plasmid contains a non-specific stuffer sequence in the cap ORF between amino acids No. 587 and No. 588 in the numbering of the wild-type AAV2 capsid protein (CAP, SEQ ID NO: 14). This helper plasmid was used in the present invention to produce novel helper plasmids for production of viral vectors with the here described tropisms.

[0030] Landegger et al., Nat Biotechnol 35, 280-284 (2017) describes a process for transducing HC.

[0031] Biining and Srivastava, Mol Ther Methods Clin Dev., Vol. 12, 2548-265 (2019) for AAV describes the CAP encoding cap gene, which codes for the viral proteins (VP) VP1, VP2, and VP2, and the assembly-activating protein (AAP). Further, integration sites for peptide inserts in viral proteins are described as being located on protruding loops of VP1, VP2 and / or VP3, e.g. amino acid 590 for AAV1, amino acid 586 for AAV3, amino acid 586 for AAV4, amino acid 575 for AAV5, amino acid 585 for AAV6, for AAV6 optionally amino acid 585 plus Y705-731F + T492V and optionally + K531E, amino acid 585 or amino acid 590 for AAV8, amino acid 589 or amino acid 588 for AAV9, in each case preferably C-terminally to the amino acid number, which refers to the wild-type CAP encoding gene. Object of the invention

[0032] It is an object of the invention to provide a pharmaceutical compound for use in the treatment of defects of the inner ear, especially a compound suitable for targeted genetic manipulation of cells of the inner ear, which compound more preferably is a viral vector.

[0033] Description of the invention

[0034] The invention achieves the object by the features of the claims, and especially by viral vectors on the basis of adeno-associated virus (AAV), preferably AAV2, for transducing cells with a nucleic acid construct contained in the viral vector for delivery of a nucleic acid construct by the viral vector, which in their capsid proteins contain an insert arranged at an integration site for a peptide insert in viral proteins which integration site is located on a protruding loop of VP1, VP2 and / or VP3. Preferably, the integration site is C-terminally adjacent to amino acid No. 587, or No. 588, or No. 453 in the numbering of the wild-type amino acid sequence of CAP (herein defined as capsid protein, which is encoded by the cap gene which contains the coding information for VP1, VP2 and VP3 which are the three viral capsid proteins forming in 1 : 1 : 10 ratio the mature capsid), which insert provides the viral vectors with specificity for targeting inner ear cells, especially for delivery of nucleic acid constructs contained in the viral vectors to inner ear cells. The viral vectors are provided for use in the treatment of defective inner ear cells, e.g. for treatment of a hearing impairment or vestibular loss, by delivering nucleic acids constructs contained in the viral vectors to inner ear cells, wherein the hearing impairment to be treated may be independent from a genetic defect or be caused by a genetic defect. Nucleic acid constructs contained in the viral vectors for delivery to inner ear cells can be set up to repair, replace or improve a genetic defect underlying the hearing impairment, support maintenance of hearing or restitution or maintenance of vestibular sense. In addition to the description and summary, the features of the viral particles and of the method of treatment are defined in the claims.

[0035] The viral vectors of the invention are based on AAV2 and comprise CAP-gene encoded proteins, herein also collectively named CAP -proteins, which are VP1, VP2 and VP3, the CAP-gene encoded proteins forming the capsid of the viral particle, which CAP-gene encoded proteins have an insert comprising or consisting of one of SEQ ID NO: 1 to SEQ ID NO: 10, preferably SEQ ID NO: 9, arranged at an integration site for an insert in AAV proteins which integration site is located on a protruding loop of VP1, VP2 and / or VP3. Preferably, the integration site is C-terminally adjacent to amino acid No. 587 or C- terminally to amino acid No. 588 or C-terminally to amino acid No. 453 of VP1, VP2 and / or VP3. Accordingly, the insert is preferably arranged between amino acids No. 587 and No. 588 in the numbering of the wild-type capsid protein (CAP, SEQ ID NO: 14). The insert generally is arranged C-terminally adjacent to amino acid No. 587 or C-terminally to amino acid No. 588 or C-terminally to amino acid No. 453 of the CAP protein, preferably C-terminally directly adjacent to amino acid No. 587 or directly adjacent to amino acid No. 588, optionally with a linker arranged N-terminally to the insert and / or optionally with a linker arranged C- terminally to the insert, and C-terminally to the insert the CAP-proteins have an amino acid section, which for VP1 is the amino acid section of No. 588 to 735, respectively of No. 589 to 735 of the wild-type capsid protein of SEQ ID NO: 14, which for VP2 is the amino acid section of No. 588 to 735, respectively of No. 589 to 735 of the wild-type capsid protein of SEQ ID NO: 14, and which for VP3 is the amino acid section of No. 588 to 735, respectively of No. 589 to 735 of the wild-type capsid protein of SEQ ID NO: 14. Preferred embodiments of the CAP comprise or consist of SEQ ID NO: 12, which is preferably encoded by SEQ ID NO: 17, wherein in SEQ ID NO: 12 the amino acids 1 to 587 are the N-terminal section and amino acids 600 to 747 (VP3), or amino acids 600 to 747 (VP2), or amino acids No. 600 to 747 (VP1) are CAP backbone, amino acids No. 588 to 590 form a linker, preferably Ala-Ser- Ala, amino acids No. 598 to 599 form a linker, preferably Ala- Ala, and amino acids No. 591 to 597 have a sequence of one of SEQ ID NO: 1 to SEQ ID NO: 10, preferably SEQ ID NO: 9. The viral vectors of the invention, when containing a nucleic acid construct, are also designated as viral vector particles, as viral vectors, or simply as vectors due to their capability for transducing the nucleic acid construct into a cell.

[0036] Accordingly, the capsid proteins of the viral vector of the invention are translated from the cap ORF which in its encoding nucleic acid between (SEQ ID NO: 15) the triplet encoding amino acid No. 587 and the triplet encoding amino acid No. 588 contains a nucleic acid insert encoding amino acids comprising or consisting of one of SEQ ID NO: 1 to SEQ ID NO: 10, preferably SEQ ID NO: 9, the insert more preferred comprising or consisting of a nucleic acid encoding a linker of two or three amino acids, e.g. Ala-Ala-Ala or preferably Ala-Ser-Ala, one of SEQ ID NO: 1 to SEQ ID NO: 10, preferably SEQ ID NO: 9, and a linker of two or three amino acids, preferably Ala-Ala. Generally herein, the order of elements of nucleic acids is described from 5' to 3', and the order of amino acid elements is described from N- terminus to C-terminus. The specific inserts of SEQ ID NO: 1 to SEQ ID NO: 10 could be identified by selecting these peptides from a random phage display library of 7-mer peptides for their binding to murine inner ear cells in in vivo experiments according to applicable ethical guidelines. In greater detail, the library of random 7-mer peptides was attached to the N-terminus of the pill protein of the filamentous Ml 3 bacteriophage and used for panning with mouse inner ear cells. The panning is based on attachment of phage to target cells due to the peptide, with washing off unbound phage for enrichment of phage that bound, then detaching the bound phage for identifying the specific 7-mer peptide of the phage. Herein, four rounds of panning in duplicate were performed with a number of 2 x 1011plaque-forming units (pfu) of the phage library on P3 organ of Corti cultures pre-treated with neomycin. The candidate 7-mer peptides encoding DNA regions of the isolated phage that were bound were sequenced using a -96 gill sequencing primer. These sequences encoded the 7-mer peptides. In addition, peptides that were identified by the panning to bind to the inner ear cells were expressed on phage and 1 x 109pfu phage were injected into the round window membrane of 2 months old female C57BL / 6 mice. 1 hour after this phage injection, temporal bones were isolated, tissue sections stained against M13 protein and subsequently visualized and photographed via fluorescent microscopy in order to demonstrate specificity of the 7-mer peptides for inner ear cells.

[0037] The following table summarizes the results of binding of the phage having in c p-encoded coat proteins the 7-mer insert:

[0038] Short description of the Figures:

[0039] - Fig. 1 schematically shows a cross-section of the inner ear,

[0040] - Fig. 2 shows a cross-section of a murine cochlea after injection of the wild-type AAV2,

[0041] - Fig. 3 shows a cross-section of a murine cochlea after injection of a viral vector of the invention containing CAP with the insert of VARI (VARI),

[0042] - Fig. 4 shows a cross-section of a murine cochlea after injection of a viral vector of the invention containing CAP with the insert of VAR2 (VAR2),

[0043] - Fig. 5 shows a cross-section of a murine cochlea after injection of a viral vector of the invention containing CAP with the insert of VAR3 (VAR3),

[0044] - Fig. 6 shows a cross-section of a murine cochlea after injection of a viral vector of the invention containing CAP with the insert of VAR4 (VAR4),

[0045] - Fig. 7 shows a cross-section of a murine cochlea after injection of a viral vector of the invention containing CAP with the insert of VAR5 (VAR5),

[0046] - Fig. 8 shows a cross-section of a murine cochlea after injection of a viral vector of the invention containing CAP with the insert of VAR6 (VAR6),

[0047] - Fig. 9 shows a cross-section of a murine cochlea after injection of a viral vector of the invention containing CAP with the insert of VAR (VAR7),

[0048] - Fig. 10 shows a cross-section of a murine cochlea after injection of a viral vector of the invention containing CAP with the insert of VAR8 (VAR8),

[0049] - Fig. 11 shows a cross-section of a murine cochlea after injection of a viral vector of the invention containing CAP with the insert of VAR9 (VAR9),

[0050] - Fig. 12 shows a cross-section of a murine cochlea after injection of a viral vector of the invention containing CAP with the insert of VARI 0 (VAR10),

[0051] - Fig. 13 A shows data of packing efficiency of a viral vector of the invention containing CAP with the insert of VAR9 (Var9) compared to wild-type AAV2 (AAV2), - Fig. 13B shows data of transduction efficiency of a viral vector of the invention containing CAP with the insert of VAR9 (Var9) compared to wild-type AAV2 (AAV2) and a synthetic viral vector described by Landegger et al. (Anc80L65),

[0052] - Fig. 13C shows data of thermal stability of a viral vector of the invention containing CAP with the insert of VAR9 (Var9) compared to wild-type AAV2 (AAV2),

[0053] - Fig. 13D shows data of uncoating efficiency of a viral vector of the invention containing CAP with the insert of VAR9 (Var9) compared to wild-type AAV2 (AAV2) and a synthetic viral vector Anc80L65 described by Landegger et al. (Anc80),

[0054] - Fig. 14A shows data for the binding affinity of a viral vector of the invention containing CAP with the insert of VAR9 (Var9) compared to wild-type AAV2 (AAV2) to heparin at different concentrations of NaCl,

[0055] - Fig. 14B shows data for the heparin competition assay on HEI-0C1 cells for a viral vector of the invention containing CAP with the insert of VAR9 (Var9) compared to wild-type AAV2 (AAV2),

[0056] - Fig. 15 A, Fig. 15B, Fig. 15C show hematoxylin and eosin staining of cochleae,

[0057] - Fig. 15D shows a scheme for the experimental procedure,

[0058] - Fig. 15E shows quantification results of survival of SGN due to ectopic expression of BDNF (brain derived neurotrophic factor) which was successfully delivered to SGN (spiral ganglion neurons) by a viral vector,

[0059] - Fig. 16A shows a scheme of the viral vector construct and location of forward (F) and reverse (R) primers used to detect vector transgene in tissues; expected PCR product size is 166 bp,

[0060] - Fig. 16B shows an agarose gel image of PCR products amplified from template plasmid in wild-type AAV2 vectors (CMV-dTomato) as control for a nucleic acid construct (lane 1), double distilled water as PCR negative control (lane 2), tissue DNA sample extracted from right (lane 3) and left (lane 4) cochlea of control (non-injected) mice, from AAV2 injected cochleae (lanes 5-7) for three mice and in contralateral cochleae (lanes 8-10), from brain tissue (lanes 11-14) and from liver (lanes 15-18) as off-target tissue,

[0061] - Fig. 16C shows an agarose gel image of PCR products amplified from template plasmid control (CMV-dTomato) contained in an AAV2 vector containing the VAR9 insert as control (lane 1), double distilled water as PCR negative control (lane 2), tissue DNA sample extracted from right (lane 3) and left (lane 4) cochlea of control (non-inj ected) mice, for three mice injected with AAV2 containing the VAR9 insert, from injected cochleae (lanes 5-7) and in contralateral cochleae (lanes 8-10), from brain tissue (lanes 11-14) and from liver (lanes 15-18) as off-target tissue

[0062] - Fig. 17A, 17C, 17E show cross-sections of the apex of murine cochleae after injection of a viral vector of the invention containing CAP with the insert of VAR9 (VAR9) with fluorescence detection for dTomato at different doses of vector particles,

[0063] - Fig. 17B, 17D, 17F show cross-sections of the middle ear of murine cochleae after injection of a viral vector of the invention containing CAP with the insert of VAR9 (VAR9) with fluorescence detection for dTomato at different doses of vector particles,

[0064] - Fig. 17G shows the result of quantification of dTomatoexpression in the apex for different doses of vector particles,

[0065] - Fig. 17H shows the result of quantification of dTomato expression in the middle ear for different doses of vector particles,

[0066] - Fig. 18A shows apex sections (Apex) and base sections (Base) of murine cochleae with histological analyses for neuronal survival of deafened mice with subsequent treatment by a viral vector containing VAR9 and an expression cassette for BDNF (VAR9-BDNF), of deafened mice without subsequent treatment (No treatment), and of unaffected healthy mice (Control),

[0067] - Fig. 18B shows apex sections (Apex) and base sections (Base) of murine cochleae with histological analyses for expression of BDNF of deafened mice with subsequent treatment by a viral vector containing VAR9 and an expression cassette for BDNF (VAR9-BDNF), and of deafened mice without subsequent treatment (No treatment),

[0068] - Fig. 18C shows the result of quantification of BDNF expression for apex and base of murine cochleae for mice, each for VAR9-BDNF treatment (VAR9-BDNF Treated Left Ear) and for the untreated right ear (Untreated Right Ear), and for unaffected healthy mice (Control).

[0069] Example 1 : Production of AAV2-based viral vectors specific for inner ear cells Oligonucleotides were synthesized which encode one of the 7-mer peptides of VARI to VAR10, flanked by a linker of alanine-serine-alanine residues at the 5’ end and a linker of two alanine residues at the 3’ end of the coding sequence for the inserted 7-mer peptide. These oligonucleotides were inserted into the CAP encoding gene of AAV2 using the AAV helper plasmid pRC99. The CAP encoding gene contains multiple open reading frames (ORFs). The cap ORF (open reading frame) encodes for the 3 AAV capsid proteins (VP1, VP2 and VP3), which assemble in a 1 :1 : 10 ratio into the 60 capsid protein subunits containing mature AAV capsid. The cap gene also encodes for the assembly activating protein (AAP) and the membrane-associated accessory protein (MAAP). The second viral gene is the rep gene encoding for the multifunctional non-structural family of Rep proteins. The Rep proteins are required for transcription regulation, genome replication and packaging. pRC99, which is described by Nicklin et al., Mol Ther 4, 174-181 (2001), encodes for both, the AAV2 rep and cap ORFs controlled by their native promoters. In pRC99, the cap gene contains a placeholder sequence at a position in the cap ORF that corresponds to the tip of the second- highest protrusion of the capsid located between N587 and R588 of SEQ ID NO: 14. Replacing the placeholder by the oligonucleotide encoding one of the 7-mer peptides with the flanking linkers in the assembled capsid results in the arrangement of the 7-mer peptide by each of the 60 protein capsid subunits at the tip of a capsid protrusion. The coding sequence for the CAP including one of the 7-mer peptides, directly flanked by the N-terminal Ala / Ser / Ala and C-terminal Ala / Ala-linkers, was confirmed by DNA sequencing.

[0070] As a representative of a therapeutic nucleic acid section, the coding sequence for enhanced green fluorescent protein (eGFP) under the control of the CMV promoter was arranged between flanking ITRs of AAV2 origin, providing a nucleic acid construct for packaging into the AAV2-based viral vectors containing capsid proteins having one of the 7-mer peptide inserts per capsid subunit. This nucleic acid construct encoding the representative of the therapeutic nucleic acid was expressed by a helper plasmid.

[0071] Viral vectors were produced in HEK293 cells and purified from cell lysates by iodixanol density gradient centrifugation by standard procedures, then isolated fractions were concentrated and further purified using Amicon centrifugal filters. Vector preparations were analyzed for content of nucleic acids as viral vector genomes per mL (vg / mL) and for content of capsids as capsid titer (cp / mL).

[0072] Analysis of viral particles gave the following results:

[0073] nd = not determined; vg = viral genome; cp = capsid

[0074] These results show that viral vector particles of the invention containing one of the inserted 7- mer flanked by Ala-Ser-Ala and Ala-Ala linkers could be produced and isolated as such, and that the viral vector particles contain the nucleic acid construct as the vector genome.

[0075] Example 2: Transducing inner ear cells by AAV2-based viral vectors

[0076] The vector variants were tested for function and general tropism by local administration in 1 to 2 months old C57BL / 6 mice. Specifically, one of the viral vector particle preparations was injected into the posterior semicircular canal. For the first analyses, differences in vector preparations regarding titers used between 9 x 107and 2 x 109vector genome containing particles in the same volume of 1 pL of each vector preparation for an injection per animal. Animals were sacrificed 7 days post injection followed by histological analysis of the cochlea. As a control, wild-type AAV2 particles containing the same nucleic acid construct encoding eGPF was used.

[0077] For an overview, Fig. 1 shows a schematic depiction of the murine cochlea, and Fig. 2 shows a representative cross-section of a murine cochlea after injection of the wild-type control viral vector (AAV2), showing essentially no expression of eGFP.

[0078] Fig. 3 shows a cross-section of a murine cochlea after injection of a viral particle containing Vari as the insert, Fig. 4 shows a cross-section of a murine cochlea after injection of a viral particle containing Var2 as the insert, Fig. 5 shows a cross-section of a murine cochlea after injection of a viral particle containing Var3, Fig. 6 shows a cross-section of a murine cochlea after injection of a viral particle containing Var4 as the insert, Fig. 7 shows a cross-section of a murine cochlea after injection of a viral particle containing Var5 as the insert, Fig. 8 shows a cross-section of a murine cochlea after injection of a viral particle containing Var6 as the insert, Fig. 9 shows a cross-section of a murine cochlea after injection of a viral particle containing Var7 as the insert, Fig. 10 shows a cross-section of a murine cochlea after injection of a viral particle containing Var9 as the insert, Fig. 11 shows a cross-section of a murine cochlea after injection of a viral particle containing Var9 (SEQ ID NO: 9) as the insert within CAP (SEQ ID NO: 13), Fig. 12 shows a cross-section of a murine cochlea after injection of a viral particle containing VarlO (SEQ ID NO: 10) as the insert within CAP.

[0079] Therein: Green: eGFP expressing cells, blue: DAPI stained nuclei, IHC: Inner hair cells, OHC: Outer hair cells, SG: Spiral ganglions, SV: Stria vascularis, MC: Marginal cells, RM: Reissner membrane, SL: Spiral ligament, DC: Deiters cells, BC: Boettcher cells, ISC: Inner sulcus cells, IDC: Interdentate cells.

[0080] The expression of eGFP in the inner ear cells shows specificity of the viral vector particles for inner ear cells as well as effective transduction of the inner ear cells for expression of a nucleic acid construct contained in and delivered by the viral vector particles.

[0081] In summary, the viral vector particles show the following biodistribution, indicating target cell specificity: IS = inner sulcus, SG = Spiral ganglion, P = Pillar, DC = Deiter, HC = Henssen, CC = Claudius, R = Root, ma = marginal, in = intermediate, Fi = Fibrocyte x: not imaged, 0+: no expression (background, 1+: low expression, 2+: intermediate expression, 3+: high expression.

[0082] Preferably, the insert for use in the treatment, especially for use in transducing preferentially inner hair cells and spiral ganglion cells has SEQ ID NO: 1 (VARI) or SEQ ID NO: 2 (VAR2) or SEQ ID NO: 5 (VAR5), the insert for use in transducing preferentially inner hair cells and spiral ganglion cells as well as cells of the stria vascularis has SEQ ID NO: 3 (VAR3), the insert for use in transducing preferentially inner hair cells and outer hair cells as well as spiral ganglion cells has SEQ ID NO: 4 (VAR4), the insert for use in transducing preferentially inner hair cells, cells of the spiral ganglion as well as cells of the stria vascularis, each at low efficiency, has SEQ ID NO: 6 (VAR6), the insert for use in transducing preferentially inner hair cells and outer hair cells as well as cells of the spiral ganglion and Deiter's cells and efficiently transducing cells of the stria vascularis has SEQ ID NO: 7 (VAR7), the insert for use in transducing preferentially inner hair cells and outer hair cells and cells of the spiral ganglion as well as Pillar cells and Deiter's cells and root cells has SEQ ID NO: 8 (VAR8), the insert for use in transducing preferentially inner hair cells and cells of the spiral ganglion as well as Pillar cells and Deiter's cells has SEQ ID NO: 9 (VAR9), the insert for use in transducing preferentially inner hair cells and outer hair cells and cells of the spiral ganglion as well as Deiter's cells, Henssen cells and Claudius cells as well as cells of the stria vascularis has SEQ ID NO: 10 (VAR10), wherein each insert optionally is arranged between linkers as described herein.

[0083] The expression of the representative eGFP from the nucleic acid construct shows the advantage of the viral vector particles to preferentially transduce distinct target cell types of the inner ear, e.g. compared to wild-type AAV2, with improved efficacy.

[0084] A preferred embodiment of the viral particle contains CAP with the insert of VAR9 (SEQ ID NO: 9, complete amino acid sequence SEQ ID NO: 13). The viral particle having the CAP with the insert of VAR9 transduces SGN in vivo with great efficiency in all turns of the cochlea. Therefore, this variant was selected for a comprehensive characterization and further proof-of-concept therapeutic application.

[0085] CAP with the insert of VAR9 was produced with efficiencies comparable to wild-type AAV2 particles, (Fig. 13 A). Thus, the insert of the 7-mer does not harm capsid assembly to produce fully functional viral vector particles. Regarding transduction efficiency on HEI-0C1 cells, a murine common progenitor cell line for sensory and supporting cells of the organ of Corti, VAR9 clearly outperformed wild-type AAV2 and also performed remarkably better compared to Anc80L65 as described by Landegger et al., Nat Biotechnol 35, 280-284 (2017), which presents the current gold standard for transducing HCs. The results shown in Fig. 13 for the characterization of the CAP with the insert VAR9 demonstrate that capsid engineering does not interfere with packaging efficiency in terms of ratio of capsid and genomic particles (Fig. 13 A). Fig. 13B shows that the CAP with VAR9 transduces HEI-OC1 cells with greater efficiency than wild-type AAV2 or than Anc80L65. Fig. 13C shows that the viral vector particle with CAP with the insert VAR9 results in decreased thermal stability of the capsid compared to wild-type AAV2. VAR9 capsids were found to disassemble at a lower temperature compared to AAV2 (60 °C and 65.8 °C, respectively, Figure 13C). Of note, despite showing a lower thermal stability, the VAR9 capsids are sufficiently stable to enable efficient vector production and purification including affinity chromatography and storage. Representative Fig. 13D of n=2 shows that the CAP with the insert of VAR9 in HEI-OC1 cells uncoates 1.5 times more efficient than wild-type AAV2, indicating a higher transduction efficiency as more vector genomes become available for transcription of the nucleic acid that was delivered by viral vector particle.

[0086] In order to analyze whether the insertion of the 7-mer peptide within CAP impairs interaction with the natural attachment receptor of AAV2, heparan sulfate proteoglycan (HSPG), the binding of heparin, a soluble analog of HSPG, to the AAV2 and the CAP-VAR9 capsid was modeled in silico, using a protein trimer as a basis. Comparing the interaction of HSPG binding residues in wild-type AAV2 and CAP with the insert of VAR9 clearly showed fewer contacts between heparin and VAR9, predicting that CAP with the insert of VAR9 binds to HSPG with lower affinity compared to wild-type AAV2.

[0087] This assumption was confirmed by heparin affinity chromatography and a heparin competition assay (Fig. 14). Firstly, the binding affinity was determined (Fig. 14A). Viral vector particles were loaded on a heparin affinity column, and flow-through (FT) and wash (WS) fraction were collected. AAVs were eluted by a salt gradient of sodium chloride (NaCl) (0.2-1.1 M) in PBS / MgCh / KCl (wash buffer with 0.137 M NaCl), and elution fractions were collected. Samples were quantified by quantitative polymerase chain reaction (qPCR).

[0088] Wild-type AAV2 vector particles were eluted from a heparin affinity column at 0.4 M NaCl. In contrast, the viral particle with capsids having the insert of VAR9 in CAP showed reduced binding with 16% of the vector genomes detected in the flow-through. Heparin-bound particles having CAP with the insert of VAR9 were eluted at a salt concentration of 0.2-0.3 M (15% and 27%, respectively), indicating a lower affinity. To confirm the lower affinity and to assay whether this has a functional consequence in cell transduction, a heparin competition assay was performed (Fig. 14B). The heparin competition assay compared the viral particles having CAP with the insert of VAR9 to wild-type AAV2 on HEI-OC1 cells (n = 2). The particles contained a nucleic acid construct encoding eGFP under the control of a CMV promoter (CMV. eGFP) and were pre-incubated with increasing heparin concentrations (0-24 U / ml) for 30 min at RT, thereafter HEI-OC1 cells were transduced with genomic particles of infection (GOI) of 500. After 48 h, eGFP expression in cell lysates was measured by flow cytometry and normalized to mock-treated AAV2 (0 U / ml heparin) transduction. In Fig. 14B, data are shown in linear scale as mean relative light units (RLU) in % of mock-treated with standard deviation. For wild-type AAV2, transduction efficiency was reduced by half (IC50) in the presence of ~1 U / ml of heparin in HEI-OC1 cells. The same concentration only slightly impaired viral particles with CAP having the insert of VAR9, which confirms the lower affinity of CAP with the insert of VAR9 to heparin compared to wild-type AAV2.

[0089] The lower binding of a viral vector particle containing CAP with a 7-mer insert, especially VAR9 (SEQ ID NO: 13) shows the advantage of viral vector particles of the invention of less binding to non-target sites, leaving more viral particles for binding to their specific target cells of the inner ear.

[0090] Viral vector particles with CAP containing the insert of VAR9 (SEQ ID NO: 13), transduce SGN in the apical and middle turns of the mature murine cochlea with doses as low as 1E7 viral vector particles. The optimal dose for SGN transduction is approximately 1E8 viral vector particles (Figure 17A-F). SGN transduction efficiency may be determined, for example, by quantifying mean fluorescence intensity in IHCs, OHCs, and SGNs, as illustrated in Figure 17G-H.

[0091] Viral vector particles with CAP containing the insert of VAR9 (SEQ ID NO: 13), which were found to efficiently transduce SGN, were produced as described above but containing a nucleic acid construct encoding for BDNF under the control of a CMV promoter.

[0092] Mice were either left untreated or treated with the ototoxic antibiotic aminoglycoside in addition with a loop diuretic for increasing the ototoxic effects. Part of the latter cohort treated with ototoxic drugs 1 week later received viral vector particles with CAP containing the insert of VAR9 and containing the nucleic acid construct encoding the expression cassette for BDNF (VAR9-BDNF). Two months post viral vector particle treatment, mice were sacrificed, followed by collection of the cochlea. Histological analysis of the cochlea revealed remarkable protective effects in all cochlear regions of mice receiving VAR9-BDNF. Fig. 15D schematically shows the experimental set-up.

[0093] Fig. 15 A, Fig. 15B, Fig. 15C, show histological analyses (scale bar = 30 pm) with hematoxylin and eosin staining, where hematoxylin stains nuclei (black) and eosin stains extracellular matrix and cytoplasm, for A control mice without ototoxic treatment and without administration of viral particles, for B ototoxic treatment by aminoglycoside and loop diuretic both without administration of viral particles, and for C ototoxic treatment by aminoglycoside and loop diuretic both with subsequent administration of viral particles AAV2 with the CAP having the insert of VAR9 (SEQ ID NO: 13) and containing the nucleic acid construct encoding BDNF at a dose of 9 x 108viral genomes.

[0094] Fig. 15E shows the quantification of neurons in basal and apical turns of the cochlea for the different treatment regimes. Significant neurodegeneration was observed in untreated mice (Figs. 15 A, 15B,) in base and apex. Neurons could be protected from degeneration in BDNF treated mice (Fig. 15C) in all turns of the cochlea.

[0095] These results provide proof for the protective effects of BDNF overexpression in SGN after HC loss in mice, wherein the overexpression of BDNF is from the nucleic acid construct contained in viral vector particles containing CAP with one of the inserts according to the invention, preferably CAP containing the insert VAR9 according to SEQ ID NO: 13. Fig. 16 shows the absence of vector genomes in the contralateral cochlea, brain and liver, and presence of vector genomes in injected cochleae for both AAV2 (Fig. 16B) and AAV2 vector containing the VAR9 insert (Fig. 16C).

[0096] In conclusion, the AAV2 vector particles containing CAP with an insert of one of VARI to VAR10, preferably an insert of VAR9, demonstrated superior properties relevant for more efficient SGN transduction, which is expected to significantly improve neural survival and to e.g. thus improve outcomes in cochlear implant recipients upon cochlear gene therapy administrating a viral vector of the invention.

[0097] Fig. 17 shows the result of lower vector doses administered on the example of AAV.VAR9. For administration of 1 x 109vg (1E9 vg), expression of dTomato is shown in the apex in Fig. 17A (Apex) and in the middle ear in Fig. 17B (Middle), showing that notable SGN transduction in the apical and middle turns of the mature murine cochlea was detected with doses as low as IxlO7vector particles (vg), although the optimal dose for SGN transduction appears to be IxlO8vector particles (lE8vg) as shown in Fig. 17C for apex, and in Fig. 17C for Middle, and for IxlO7vector particles (lE7vg) as shown in Fig. 17E for apex, and in Fig. 17F for Middle. Quantification was based on mean fluorescence measurements of IHCs, OHCs, and SGNs as shown in Fig. 17G for Apex and Fig. 17H for Middle.

[0098] Viral vector particles with CAP containing the insert of VAR9 (SEQ ID NO: 13), which were found to efficiently transduce SGN, were produced as described above but containing a nucleic acid construct encoding for BDNF under the control of a CMV promoter.

[0099] Mice were either left untreated or treated with the ototoxic antibiotic aminoglycoside in addition with a loop diuretic for increasing the ototoxic effects. Part of the latter cohort treated with ototoxic drugs 3 months later received viral vector particles with CAP containing the insert of VAR9 and containing the nucleic acid construct encoding the expression cassette for BDNF (VAR9-BDNF). Another three months post viral vector particle treatment, mice were sacrificed, followed by collection of the cochlea. Histological analysis of the cochlea revealed remarkable protective effects in all cochlear regions of mice receiving VAR9-BDNF. Results are shown in Fig. 17 for expression of dTomato as representative transgene, and in Fig. 18 for expression of BDNF. Fig 18A shows histological analyses for neuronal survival with Tuj 1 antibody staining for residual SGN. Normal SGN morphology was preserved in healthy control mice (right, Control). SGN degeneration could be observed in the ototoxic drug treated, viral vector- untreated cohort six months post-deafening with a few surviving neurons seen in the untreated contralateral SGN (center, No treatment). Degeneration is more marked in the untreated basal turn with degeneration of all neurites but a few surviving neuronal cell bodies (center). SGN could be protected from degeneration in the ototoxic drug treated, viral vector-treated cohort (left, VAR9-BDNF) to a similar extent to healthy control levels (Control).

[0100] Fig. 18B shows histological analyses to confirm BDNF expression in the cochlea of the ototoxic drug treated, viral vector-treated cohort (left, VAR9-BDNF) and ototoxic drug treated, viral vector-untreated cohort (right, No treatment). Minimal BDNF immunofluorescent signal was detected on the viral vector-untreated control ear, whereas VAR9-BDNF treatment resulted in BDNF expression in SGNs. This confirms the successful delivery of BDNF -encoding vector genomes by VAR9 and subsequent expression of BDNF in the targeted cell population.

[0101] Fig. 18C shows quantification of SGN from histological results of Fig. 18A and confirms significant protective effects in basal and apical cochlear turns of mice receiving VAR9- BDNF (blue), to healthy control levels (green) as compared to ototoxic drug treated mice (red).

[0102] These results show that BDNF gene transfer with AAV.Var9 protects from SGN degeneration in deafened mice. In this experiment, mice were deafened with a combination of an aminoglycoside and loop diuretic resulting in loss of all residual hearing. Part of this cohort received AAV.VAR9-BDNF (9x 108vg per inner ear) three months after artificial deafening. Three months after AAV. VAR9-BDNF treatment, mice were euthanized followed by collection of cochleae. To assess tissue morphology of SGN, histopathological analysis of “deafened AAV. Var9-BDNF treated” and “deafened, vector-untreated” and “untreated wildtype control” cochleae was performed by immunostaining residual SGNs with TuJl antibody. Normal SGN morphology was preserved in healthy control mice as expected (Fig. 18 A). However, immense SGN degeneration could be observed in the “deafened, vector- untreated cohort” six months post-deafening with a few surviving neurons seen in the untreated contralateral spiral ganglion. Degeneration is more marked in the untreated basal turn with degeneration of all neurites but a few surviving neuronal cell bodies (Fig. 18 A). Quantification of SGN confirmed the significant protective effects in basal and apical cochlear turns of mice receiving AAV. VAR9-BDNF (1) to healthy control levels (3) as compared to deafened untreated mice (2) (Fig. 18C). To confirm BDNF expression in the cochleae, histological analysis of “deafened, vector-untreated” and “deafened, AAV. Var9- BDNF -treated” cochleae (Fig. 18B) were performed. As expected, minimal BDNF immunofluorescent signal was detected on the untreated control ear, whereas AAV. VAR9- BDNF treatment resulted in BDNF expression in SGNs (Fig. 18C). This confirms the successful delivery of BDNF -encoding vector genomes by AAV. Var9 and subsequent expression of BDNF in the targeted cochlear regions following PSCC administration of AAV.VAR9.

Claims

Claims1. Viral vector based on AAV for use in the treatment of a defect of the inner ear, the viral vector having a capsid formed of CAP-gene encoded proteins having an insert between amino acids No. 587 to 588 in the numbering of the wild-type CAP-gene encoded protein, or between amino acids No. 588 to 589 in the numbering of the wildtype CAP-gene encoded protein, or between amino acids No. 453 to 454 in the numbering of the wild-type CAP-gene encoded protein, the insert being selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 10.

2. Viral vector for use in the treatment according to claim 1, wherein the AAV is AAV2, and the wild-type CAP-gene encoded protein (CAP) has SEQ ID NO: 14.

3. Viral vector for use in the treatment according to one of the preceding claims, containing a nucleic acid construct encoding at least one gene selected from the group of Ushlc, GJB2, MY07, Atohl, BDNF, STRC, LoxHDl, EPS8, GIPC3, CDC14A, MSRB3, DCDC2, PNPT1, TMEM132E, CDH23, SLC26A4, FAM65B, MY03A, CLIC5, HGF, USH2A, TBC1D24, MET, OTOF, PCDH15.

4. Viral vector for use in the treatment according to one of the preceding claims, wherein the treatment is treatment of defects or impairments of supporting cells (SCs) which are present on the basilar membrane of the inner ear, of defects or impairments of the inner hair cells (IHCs) or of the outer hair cells (OHCs) of the inner ear, of defects or impairments of cells of the Stria vascularis of the inner ear, of defects or impairments of spiral ganglion neuron (SGN) cells, of tinnitus and / or peripheral vestibular loss, and / or the treatment is for restitution of hearing or maintenance of hearing, or for restitution or maintenance of vestibular sense.

5. Viral vector for use in the treatment according to one of the preceding claims, wherein between the insert of one of SEQ ID NO: 1 to SEQ ID NO: 10 and amino acid No.587 or No. 588 or No. 453 a linker of two to three amino acids is arranged, and / or wherein between the insert of one of SEQ ID NO: 1 to SEQ ID NO: 10 and amino acid No. 588 or No. 589 or No. 454 of the wild-type capsid protein, a linker of two to three amino acids is arranged.

6. Viral vector for use in the treatment according to one of the preceding claims, wherein a linker of at least two amino acids is arranged N-terminally to the insert of one of SEQ ID NO: 1 to SEQ ID NO: 10, and / or a linker of at least two amino acids is arranged C-terminally to the insert of one of SEQ ID NO: 1 to SEQ ID NO: 10.

7. Viral vector for use in the treatment according to one of the preceding claims, wherein the viral vector in its capsid contains a nucleic acid construct encoding a gene of a protein or regulatory RNA or a gene editing construct consisting of promoter, geneediting tool, such as but not limited to CRISPR-Cas systems including e.g. nucleases or nickases and guide RNAs, base editors, or prime editors, optionally donor template, optionally a reporter gene, or a combination of at least two of these, each of which having supporting activity in the restitution of hearing or maintenance of hearing.

8. Viral vector for use in the treatment according to one of the preceding claims, wherein the nucleic acid construct contains a gene selected from the group of Ushlc, GJB2, MY07, Atohl, BDNF.

9. Pharmaceutical product for treatment of defects of the inner ear, the product comprising a viral vector based on AAV according to one of the preceding claims.

10. Method of treatment of a defect of the inner ear, comprising administration of a viral vector according to one of claims 1 to 8 or administration of a pharmaceutical product according to claim 9.

11. Process for producing a viral vector for use in the treatment of a defect of the inner ear, the process comprising expressing capsid proteins from a nucleic acid containing the cap ORF which in its encoding nucleic acid between the triplet encoding amino acid No. 587 and the triplet encoding amino acid No. 588 of SEQ ID NO: 14, or between the triplet encoding amino acid No. 588 and the triplet encoding amino acid No. 589 of SEQ ID NO: 14, or between the triplet encoding amino acids No. 588 to 589 in the numbering of the wild-type CAP-gene encoded protein of SEQ ID NO: 14 contains a nucleic acid insert encoding amino acids comprising or consisting of one of SEQ ID NO: 1 to SEQ ID NO: 10.

12. Process according to claim 11, wherein the AAV is AAV2, and the wild-type CAP- gene encoded protein (CAP) has SEQ ID NO: 14.

13. Process according to one of claims 11 to 12, the viral vector containing a nucleic acid construct encoding at least one gene selected from the group of Ushlc, GJ 2, MY07, Atohl, BDNF, STRC, LoxHDl, EPS8, GIPC3, CDC14A, MSRB3, DCDC2, PNPT1, TMEM132E, CDH23, SLC26A4, FAM65B, MY03A, CLIC5, HGF, USH2A, TBC1D24, MET, OTOF, PCDH15.

14. Process according to one of claims 11 to 13„ wherein the treatment is treatment of defects or impairments of supporting cells (SCs) which are present on the basilar membrane of the inner ear, of defects or impairments of the inner hair cells (IHCs) or of the outer hair cells (OHCs) of the inner ear, of defects or impairments of cells of the Stria vascularis of the inner ear, of defects or impairments of spiral ganglion neuron (SGN) cells, of tinnitus and / or peripheral vestibular loss, and / or the treatment is for restitution of hearing or maintenance of hearing, or for restitution or maintenance of vestibular sense.

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