Patent Application
20240191257
The invention relates to gene therapy vectors for treating, preventing or reversing retinal degeneration.
CDHR1 is essential for outer segment disc morphogenesis in the mammalian retina. CDHR1-associated retinal degeneration is a recessively inherited human disease for which there is no effective treatment. Biallelic null variants in CDHR1 result in mis-stacking of the outer segment discs of rod and cone photoreceptors, with early rod and cone dysfunction associated with shortened photoreceptor outer segments and progressive photoreceptor cell death. This phenotype is recapitulated by the Cdhr1−/− murine model. Progressive photoreceptor cell death results in legal blindness in all affected patients before the age of 60. Patients with CDHR1-associated retinal degeneration experience both rod and cone photoreceptor degeneration, which means that they lose both their peripheral vision/visual field and also their central reading/colour vision which is normally preserved until late in many other forms of retinitis pigmentosa. This makes CDHR1-associated retinal degeneration visually debilitating. CDHR1-associated retinal degeneration is also relatively common: the global genetic prevalence is estimated at ˜211,832 affected individuals worldwide, including ˜62,665 individuals in Europe and North America. Biallelic hypomorphic variants in CDHR1 have also recently been identified as one of the main genetic causes of macular dystrophy.
CDHR1 encodes a photoreceptor-specific cadherin. Cadherins form a superfamily of proteins characterised by the presence of two or more extracellular cadherin repeats and serve crucial roles in cell-to-cell adhesion. In highly evolved sensory cells, such as the photoreceptors in the retina and the hair cells of the inner ear, cadherins serve more complex functions, principally through heterophilic interactions with other proteins to support the function of the cilia and outer segment. The cilia is crucial in that that links photoreceptor inner segments, where proteins are manufactured, to the outer segment discs which are highly specialised structures that support the process of phototransduction (i.e. conversion of light energy into electrical potentials). The heterophilic binding partner of CDHR1 in the inner segment is unknown.
Methods are being developed to deliver gene therapy to the cells of the retina using adeno-associated virus (AAV) (MacLaren et al. (2016), American Academy of Ophthalmology 123(10) Suppl. S98-S106; Fischer M D et al. (2017), Molecular Therapy. 25 (8): 1854-1865). However, membrane-bound proteins, such as cadherins, are not usually considered attractive targets for gene therapy as it is presumed they are more likely to evoke undesirable immune responses. To date no transgene expressing a cadherin has been shown to ameliorate any disease phenotype in vivo in any organ.
The inventors have undertaken longitudinal deep phenotyping of the Cdhr1−/− mouse model to demonstrate the extent of mimicry of the human phenotype and identify suitable outcome measures to assess downstream gene rescue. They have designed vector constructs that efficiently express CDHR1 protein in the photoreceptor cells of the Cdhr1−/− mouse retina. Treatment results in significant and unexpected long-term improvement of retinal function in both rod- and cone-specific responses. Surprisingly, rod and cone rescue was achieved through the prevention of photoreceptor cell death and, even more unexpected, regeneration of photoreceptor outer segments. In other words, treatment not only slowed or prevented further retinal degeneration, it also improved the cellular structure and function of the retina, reversing previous degeneration. AAV8.CDHR1 gene therapy restored outer retinal structure in the Cdhr1−/− mouse model with restoration of the ellipsoid zone and photoreceptor outer segments as seen on optical coherence tomography imaging. This finding could explain the functional improvements since the photoreceptor outer segment houses key proteins (such as rhodopsin) which mediate phototransduction (i.e. convert light energy into electrical potentials).
Accordingly, in a first aspect the invention provides a method of treating, preventing or reversing retinal degradation in a subject in need thereof, the method comprising administering to the subject a vector that expresses a Cadherin-related family member 1 (CDHR1) polypeptide.
In a further aspect the invention provides gene therapy vector that expresses a CDHR1 polypeptide.
In a further aspect the invention provides host cell that produces the gene therapy vector.
In a further aspect the invention provides method for production of the gene therapy vector, the method comprises providing a host cell as described above and culturing the host cell under conditions suitable for the production of the vector.
In a further aspect the invention provides a pharmaceutical composition comprising the gene therapy vector.
In a further aspect the invention provides the gene therapy vector or the pharmaceutical composition for use in a method of treating, preventing or reversing retinal degeneration.
In a further aspect the invention provides the use of the gene therapy vector according in the manufacture of a medicament for of treating, preventing or reversing retinal degeneration in a subject.
The disclosure 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.
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 encode a therapeutic gene product, a CDHR1 polypeptide, and can be used to express the product in photoreceptor cells in the retina. The vector may be of any suitable type, such as a plasmid vector or a minicircle DNA. Typically, the vector is a viral vector. The viral vector may, for example, be an adeno-associated virus (AAV), a retrovirus, a lentivirus, a herpes simplex virus, or an adenovirus. Relevant sections of the description relating the AAV derived vectors also apply in the case of vectors derived from other sources, such as those discussed further below.
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 replication-deficient.
The AAV genome may be in single-stranded form, either positive or negative-sense, 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, p19 and p40 promoters (Laughlin et al., 1979, PNAS, 76:5567-5571). For example, the p5 and p19 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, CDHR1. 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 or AAV9.
The capsid may also be derived from any suitable AAV serotype, such as AAV8. 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:
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 a CDHR1 polypeptide 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 a CDHR1 transgene and 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 a transgene polypeptide (CDHR1) 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 double-stranded 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: 1 (5′ITR) and/or 9 (3′UTR) or variants having at least 80% or 85%, or 90%, or 95% or 98% or 99% sequence identity to SEQ ID NOs: 1 or 9, or up to 1, 2, 3, 4 or 5 insertions, deletions, or substitutions in the amino acid sequences of SEQ ID NO: 1 or 9.
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.
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 co-receptor 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 self-priming 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.
The vector may comprise a retrovirus genome or a derivative thereof. Derivatives of a retrovirus genome include any truncated or modified forms of a retrovirus genome which allow for expression of a CDHR1 transgene from the vector in vivo in accordance with the present invention.
As with AAV derived vectors, a retrovirus derived vector will typically comprise a derivative of a retroviral genome comprising the minimal viral sequences required for packaging and subsequent integration into a host. For retrovirus derived vectors, one or more long terminal repeats (LTRs) are the minimum element required for replication and packaging of the vectors and subsequent integration into the target cell to provide permanent transgene expression. However, other elements may also be present. For example, a human immuno deficiency virus (HIV) derived vector will typically comprises the HIV 5′ LTR, which is necessary for integration into the host cell genome, the Psi signal, which is necessary for packaging of viral RNA into virions, a promoter for the transgene, and the 3′ LTR. Other suitable retroviral vectors may for example be derived from murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), and combinations thereof.
The tropism of a retrovirus derived vector is determined by the viral envelope proteins. Targeting of the appropriate cells, for example photoreceptor cells or RPE cells of the retina, may be enhanced by incorporating ligands for the target cells into the viral envelope.
The vector may comprise an adenovirus genome or a derivative thereof. Derivatives of an adenovirus genome include any truncated or modified forms of an adenovirus genome which allow for expression of a CDHR1 transgene from the vector in vivo in accordance with the present invention.
A large number of human adenoviral serotypes have been identified and they are categorized into six subgenera (A through F) based on nucleic acid comparisons, fibre protein characteristics, and biological properties. For example, group A includes serotypes 12 and 31, group B includes serotypes 3 and 7, group C includes serotypes 2 and 5, group D includes serotypes 8 and 30, group E includes serotype 4, and group F includes serotypes 40 and 41.
The core of an adenovirus virion contains the linear double-stranded DNA genome and associated proteins V, VII, X (mu), IVa2, and terminal protein (TP). The genome organization of different adenoviruses is conserved and has been proposed to have a timing function, wherein the ends of the genome are transcribed first (the immediate early genes E1 and E4 are located at opposite ends of the linear genome). Early transcription of E1 and E4 leads to the opening of the central region of the genome, allowing transcription of the central region.
Adenoviral genomes typically comprise eight RNA polymerase II transcriptional units: five early units, E1A, E1B, E2A-E2B, E3, and E4; two delayed early units, IX and IVa2; and the Major Late transcriptional unit. The Major Late transcriptional unit is further subdivided into L1-L5 regions based upon the use of alternative splicing sites. The transcriptional units often express proteins of similar function. For example, the E1A unit codes for two proteins responsible for activation of transcription and induction of S-phase upon cellular infection; the E1B transcription unit encodes two proteins that inhibit cellular apoptosis; the E3 transcriptional unit is involved in evasion of the immune response; and the Major Late transcriptional unit encodes structural proteins necessary for assembly of the capsid.
Heterologous transgene sequences may be inserted into adenoviral genomes, for example in the early transcriptional units and in the coding regions of various structural proteins, such as hexon, penton, and fiber. Deletions may have been made in the adenoviral genome (e.g., in the El regions) to create replication-defective adenoviral vectors, which have generally been considered safer for administration to human subjects.
In the present invention, the adenovirus may be any adenovirus or derivative suitable for delivery of the transgene to target cells. The adenovirus may be any serotype but is typically Ad5 or Ad2. An adenovirus derived vector of the invention may comprise all or part of the genome of any adenoviral serotype, as well as combinations thereof (i.e., hybrid genomes).
The adenoviral vector used in the invention may be either replication incompetent or replication competent. Such vectors are well known. For example, in a replication incompetent vector the E1 region may be deleted and replaced with an expression cassette with an exogenous promoter driving expression of the heterologous transgene. Usually, the E3 region is also deleted. Deletion of E3 allows for larger inserts into the E1 region. Such vectors may be propagated in appropriate cell lines such as HEK 293 cells which retain and express the E1A and E1B proteins. Other vectors also lack the E4 region, and some vectors further lack the E2 region. E2 and E4 vectors must be grown on cell lines that complement the E1, E4 and E2 deletions.
Vectors may also be helper dependent vectors, which lack most or all of the adenoviral genes but retain cis-acting sequences such as the inverted terminal repeats as well as packaging sequences that are required for the genome to be packaged and replicated. These vectors are propagated in the presence of a helper adenovirus, which must be eliminated from the vector stocks. Once again, such systems are well known in the art.
The capsid is composed of seven structural proteins: II (hexon), III (penton), IIIa, IV (fiber), VI, VII, and IX. The capsid comprises 252 capsomeres, of which 240 are hexon capsomeres and 12 are penton capsomeres. Hexon capsomeres, which are trimers of the hexon protein, make up about 75% of the protein of the capsid. Penton capsomeres, which are pentamers of the penton protein, are situated at each of the 12 vertices of the virion. Each penton capsomer is bound to six adjacent hexon capsomeres and a fiber. The fiber, which is usually a trimer of the fiber protein, projects from the penton capsomer. The hexon protein and, to a lesser extent, the fiber protein comprise the main antigenic determinants of an adenovirus and also determine serotype specificity.
An adenovirus derived vector is particularly suitable for use when a transient expression of a transgene is preferred.
The vector may comprise an herpes simplex virus (HSV) genome or a derivative thereof. Derivatives of an HSV genome include any truncated or modified forms of a HSV genome which allow for expression of a CDHR1 transgene from the vector in vivo in accordance with the present invention.
Herpes simplex virus (HSV) naturally establishes a life-long latent infection of human peripheral sensory neurons. Recombinant HSV vectors are genetically modified to be incapable of replication, but establish a latent-like state in neurons in vitro and in vivo.
The term “CDHR1 polypeptide” as used herein encompasses any wildtype human CDHR1 as expressed by a healthy subject not having or expected to develop a CDHR1-associated retinal degeneration, for example CDHR1 having the amino acid sequence of SEQ ID NO: 6. The term also encompasses functional variants of such wildtype CDHR1, that is any variant that retains the normal function(s) of CDHR1 in vivo. CDHR1 appears to function in the development of nascent outer segment discs, possibly assisting in their horizontal elongation through connections with the periciliary ridge of the inner segment. There is then a cleavage event which likely severs the connections between CDHR1 and the inner segment binding partner (currently unidentified). Is has been proposed that ADAM10 may be this catalyst, although this has not been verified. ADAM10 is regulated by Sfrp1 (PMID: 32198470). Overall, CDHR1 functions in outer segment disc morphogenesis in order to produce regularly stacked outer segment discs. It is not known to have any other function. The normal function of a CDHR1 polypeptide could, for example, be tested in the Cdhr1−/− mouse model, for example with use of immunohistochemistry, western blot, ERG testing, or a combination of these tests.
In some cases, the CDHR1 polynucleotide may be a variant of the polynucleotide sequence of SEQ ID NO: 6 comprising 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. In some cases, the CDHR1 sequence may also have additional sequence elements or tags at the 5′ or 3′ end. 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.
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 (i.e., % identity=number of identical positions/total number of positions in the reference sequence×100).
Typically the sequence comparison is carried out over the length of the reference sequence, for example, for a CDHR1 polypeptide, SEQ ID NO: 6. If the sequence is shorter than SEQ ID NO: 1, the gaps or missing positions should be considered to be non-identical positions.
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 above. 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.
Wildtype CDHR1 has 6 extracellular cadherin repeat domains, each of which contain calcium binding sites which appear crucial for its function (SEQ ID NOs: 13 to 18). The extracellular domains of CDHR1 are highly conserved which supports an important biological function. These may include binding sites for interacting partners (such as the inner segment binding partner, and the enzyme which cleaves the Cdhr1 ectodomain as shown by Rattner et al, 2004). The cytoplasmic domain is less conserved. Accordingly, in some cases the CDHR1 polypeptide used in the present invention comprises at least one, or at least 2, 3, 4, 5 or all 6 of the cadherin repeat domains corresponding to SEQ ID NOs: 13 to 18, or corresponding to SEQ ID NOs: 13 to 18 except for a small number of conservative changes as described above.
In some cases, the CDHR1 polypeptide is a codon-optimised version of the CDHR1 polypeptide of SEQ ID NO: 6, or of any other suitable CDHR1 polypeptide described herein.
A polynucleotide sequence encoding a CDHR1 polypeptide is any sequence which encodes such a CDHR1 polypeptide as described above. For example, vectors comprising a codon-optimised version of the nucleotide sequence of SEQ ID NO: 5 that encodes the same CDHR1 polypeptide of SEQ ID NO: 6 are explicitly encompassed within the scope of the present invention. In some cases the vector comprises the polynucleotide sequence of SEQ ID NO: 5.
In the vector, the nucleic acid encoding the transgene product, i.e. the CDHR1 polypeptide, is typically operably linked to a promoter. 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 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: 2, or the human rhodopsin promoter, which may have the sequence of SEQ ID NO: 3, or functional variants thereof. One or more other regulatory elements, such as enhancers, postregulatory 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. 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.
A vector of the invention may typically comprise the following elements in a 5′ to 3′ direction: (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: 1, 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:1; (b) a promoter sequence, for example any promoter described herein, wherein the promoter is operably linked to a sequence encoding the CDHR1 polypeptide, for example the GRK1 promoter comprising the sequence of SEQ ID NO: 2; (c) a translation initiation sequence, such as the Kozak consensus sequence GCCACC; (d) optionally a chicken beta-actin promoter exon-intron-exon sequence (Ex/In/Ex), such as the sequence of SEQ ID NO: 4, 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: 4; (e) a sequence encoding the CDHR1 polypeptide, such as the sequence of SEQ ID NO: 5; (f) optionally a woodchuck hepatitis post-transcriptional regulatory element (WPRE) having the sequence of SEQ ID NO: 7, 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: 7; (g) a polyadenylation tail sequence, such as the bovine growth hormone polyadenylation tail sequence of SEQ ID NO: 8; and (h) 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: 9, 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:9. These components (a) to (h) ((d) and (f) being optional, i.e. each independently either present or absent) may be referred to as an expression cassette.
There may be intervening sequences between the some or all of the different components (a) to (h). 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.
In some cases the vector comprises the sequence of any one of SEQ ID NOs: 10, 11 and 12. In some cases the vector has the sequence of SEQ ID NO: 10 or SEQ ID NO: 12, or variants thereof as set out below. In some cases the vector comprises the expression cassette (components (a) to (h)) within SEQ ID NOs: 10, 11 or 12, but a different vector backbone to that described in the Examples herein. For example, a different antibiotic resistance gene, such as kanamycin resistance gene, could be used in place of the ampicillin resistance gene. In some cases the vector comprises the expression cassette of any one of the vectors of SEQ ID NOs: 10, 11 and 12, except that one of the components (a) to (h) (or two or three or four of the components) is a variant of the component present in the reference sequence SEQ ID NOs: 10, 11 and 12 as set out above. For example, the vector may have the expression cassette of SEQ ID NO: 10, except that component (a) is a variant of the 5′ITR of SEQ ID NO: 1, having at least 70%, or 80% or 85% or 90% or 95% or 98% or 99% sequence identity to SEQ ID NO:1. In some cases the vector comprises a sequence having at least 70%, or at least 80%, or at least 85% or 90% or 95% or 98% or 99% or 99.6%, or 99.9% sequence identity to SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, optionally in combination with any of the sequence variation restraints set out above. For example, in one embodiment, the vector may comprise a sequencing having at least 95% sequence identity to the vector of SEQ ID NO: 10 or SEQ ID NO: 12, but each of components (a), (b), (c), (e), (g) and (h) is individually has at least 98% or 99% sequence identity to the corresponding component of SEQ ID NO: 10 or 12. In another example, the vector may have at least 95% sequence identity to the vector of SEQ ID NO: 10 or SEQ ID NO: 12, but comprise the full sequence of the expression cassette of SEQ ID NO: 10 or SEQ ID NO: 12, except that a component (a) is a variant of the 5′ITR of SEQ ID NO:1, having at least 70%, or 80% or 85% or 90% or 95% or 98% or 99% sequence identity to SEQ ID NO:1.
A vector of the invention may be prepared by standard means known in the art for provision of vectors for gene 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 PA317 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, i.e. a CDHR1 transgene. 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.
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 biallelic variants in CDHR1. No current treatment exists for such patients, with inevitable blindness. Such methods of treatment form part of the present invention, as does use of the vectors and 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 CDHR1-associated retinal degeneration or retinal dystrophy, or any condition associated with a loss of function of CDHR1 in a subject or patient. In some cases the subject may have biallelic null mutants of CDHR1. Examples are provided in Example 9 and Table 2. In other cases, the subject may have a one or more hypomorphic alleles of CDHR1. Conditions that may be treated include CDHR1-associated 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 sequence variants in the CDHR1 gene.
In some cases, the treatment achieves or is intended to achieve any one or more of the following effects:
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.
A particularly surprising result of the mouse model treatment described herein was the ability of the CDHR1-expressing vectors to reverse structural degeneration and the magnitude of functional responses seen at early timepoint in AAV-treated Cdhr1−/− mice. For example, the improvement in A-wave responses following high-dose AAV injection was of a greater effect size than seen at the equivalent time-point in other pre-clinical proof-of-concept studies, such as Rpgr gene therapy (ref: PMID: 28549772). Moreover the beneficial effects appeared to increase over time as the degeneration progressed in untreated eyes. A sustained therapeutic effect is further supported by the surprising finding of preserved retinal thickness measurements in AAV-treated Cdhr1−/− mice for 12 months following treatment. Detailed natural history studies using OCT imaging in Cdhr1 mice identified that relatively severe early functional deficits (affecting both rod and cones) was associated with poorly formed photoreceptor outer segments to which we attributed the early functional deficits; further functional losses were attributed to progressive photoreceptor cell death. Preservation of outer segments was therefore a key therapeutic goal following AAV gene therapy, since it is one of the key morphological characteristics of Cdhr1−/− histological sections and human CDHR1-associated retinal degeneration. Importantly, restoration of the photoreceptor outer segments following AAV gene therapy was entirely unexpected and the most surprising of all demonstrated beneficial effects, since it implies restoration of the key function of Cdhr1—the morphogenesis of photoreceptor outer segments. These effects have not to our knowledge been shown following gene therapy in any pre-clinical model of retinal degeneration. Furthermore, the photoreceptor outer segments continued to regenerate between 1- and 6-months post-injection within the treated retina; both in terms of the reflectivity of the band representing outer segments, and in the extent of the regeneration. Moreover, the ellipsoid zone, formed of the junction of inner segment and outer segment boundaries was most hyperreflective at 12-months, with preservation of the regenerated outer segments (
These observations are remarkable as they suggest sustained expression of Cdhr1 within episomes in photoreceptor cells, correct trafficking of Cdhr1 to the base of the photoreceptor outer segments, appropriate post-translational modification that permits Cdhr1 to interact with at least two as yet unconfirmed interacting molecules (the inner segment binding partner which permits horizontal outer segment disc elongation and the catalyst that cleaves its ectodomain, allowing outer segment discs to grow outwards towards the RPE). Furthermore, all of these beneficial molecular responses have been achieved through the expression of a cadherin cell surface molecule that may be considered an unattractive target for gene therapy given that Cdhr1 is a membrane-bound protein. However, at equivalent doses, we showed no toxicity in either wildtype or Cdhr1−/− retinae.
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, 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.
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. 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. More specifically 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 108, or 109 or 2.5×109 or 5×109; and 1015, or 1014, or 1013, or 1012, 1011 or 5×1010 or 2.5×1010 or 1010 viral genomes (vg), or any range thereof, such as 2.5×109 to 5×1010 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. However, extensive dosing studies of vectors expressing the CDHR1 transgene were performed in Cdhr1−/− and C57BL6J (wildtype mice). These data suggest a therapeutic dosing range, for example for GRK1.CDHR1.pA in the Cdhr1−/− mouse. In the case of CDHR1, the finding of therapeutic efficacy at a dose of 1.5×108 is a log unit less than was required to show a therapeutic benefit in a mouse model of RPGR-related retinitis pigmentosa (PMID: 28549772). A dose of 2×109 of AAV8.RPGR was required to show therapeutic benefit in the Rpgr mouse model. Benefit was shown in phase 1 clinical trials with the resulting vector (PMID: 32094925) with doses ranging from 5×1010-2.5×1011. An approximation suggests that a dosing range of 5×109-2.5×1010 may be sufficient to show a therapeutic benefit in patients with CDHR1-associated retinal degeneration.
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)
The vectors, pharmaceutical compositions or other products of the invention as described herein can be packaged into a kit. The kit may additionally comprise suitable means for administering the product and/or instructions for use. Such kits are an aspect of the present invention.
Retinal imaging studies of Cdhr1 mutant mice have never previously been undertaken. The objectives of retinal imaging studies in Cdhr1 mutant mice and experimental results pertaining to that objective are presented below.
The full-length human CDHR1 coding sequence (NCBI transcript ID: NM_033100; 2,580 bp) was amplified by KOD polymerase chain reaction, and subcloned into a vector backbone containing the human rhodopsin kinase promoter (GRK1, 199 bp), a Kozak consensus sequence (GCCACC) for translation initiation in the 5′ position, and a downstream bovine growth hormone polyadenylation tail for the stabilisation of mRNA transcripts (GRK1.CDHR1.pA). A second plasmid construct was created in which cis-acting enhancing sequences were added; the exon-intron-exon sequence from the chicken beta-actin promoter cloned in following the GRKI promoter and the woodchuck hepatitis post-transcriptional regulatory element (WPRE) downstream of the CDHR1 coding sequence (GRK1.In.CDHR1.WPRE.pA). A third plasmid construct omitted the exon-intron-exon sequence, but the unenhanced (A), and the maximally enhanced (C) constructs were packaged into AAV for downstream assessments. A schematic representation of the three vector construct designs containing the human CDHR1 transgene are shown in
The CDHR1 expressing constructs were sequence verified, amplified and purified. Two of the three constructs, were selected for production of experimental AAVs (p.AAV8.GRK1.CDHR1.pA and p.AAV8.GRK1.In.CDHR1.WPRE.pA), being packaged into wildtype AAV8 capsids (PlasmidFactory, Bielefield, Germany) following culture in HEK293T cells, lysis and isolation of viral particles using an iodixanol gradient, and purification. SDS-polyacrylamide gel electrophoresis confirmed viral purity through the presence of AAV viral capsid proteins (VPN1, VPN2, VPN3) without contaminants (FIG. 15). AAV titre was achieved using quantitative polymerase chain reaction (qPCR) with primers directed against the CDHR1 transgene; achieved viral titres were 8.95×1012 for GRK1.CDHR1.pA and 2.97×1012 for GRK1.In.CDHR1.WPRE.pA (
In the absence of a stable photoreceptor cell line, expression of CDHR1 was demonstrated in vivo in the Cdhr1−/− knockout mouse line. The AAV8.GRK1.CDHR1.pA and AAV8.GRK1.In.CDHR1.WPRE.pA vectors were delivered by subretinal injection at a dose of 1.5×109 with vehicle control (PBS-0.001% PF68) of the same volume delivered to the fellow eye to control for the effect of surgical retinal detachment (experimental design summarised in
The maximum tolerated sub-retinal dose of each AAV8.CDHR1 vector was determined using outer retinal thickness measurements on optical coherence tomography (OCT) imaging 4-weeks after intervention (experimental design is summarised in
In Cdhr1−/− mice, the maximum tolerated dose as evaluated by retinal thickness measurements 4-weeks following subretinal injection was 7.5×108 gc (
The inclusion of two cis-acting enhancing elements led to a higher degree of measured toxicity when compared to the unenhanced vector, as determined by retinal thickness measurements post-injection (
In C57BL6J mice, subretinal delivery of 7.5×106 gc to 1.5×107 gc did not demonstrate retinal thinning versus PBS-injected control eyes (P>0.95 for both doses, two-way ANOVA). In Cdhr1−/− mice, a dose of 1.5×107 gc did not produce more retinal thinning than seen in PBS-injected control eyes (P=0.32, two-way ANOVA), although this was seen at doses of 7.5×107 gc or higher. Although a safe dosing limit was thus identified, time constraints led to the selection of AAV8.GRK1.CDHR1.pA for a longitudinal gene rescue experiment.
A prospective, open-label, paired controlled trial was undertaken to evaluate the safety and efficacy of the AAV8.GRK1.CDHR1.pA vector on retinal structure and function in Cdhr1−/− and C57BL6J mice (experimental design is shown in
Animals in both Cdhr1−/− and C57BL6J groups underwent sub-retinal injection of AAV8.GRK1.CDHR 1.pA with PBS vehicle control in the fellow eye at 3-4 weeks of age (post-weaning). The subretinal injection volume was sufficient to detach the superior hemi-retina, leaving the inferior retina attached as an internal control for retinal imaging studies which can evaluate both retinal locations. OCT imaging was undertaken at 4-weeks post-injection to determine the effect of surgically induced retinal detachment on outer retinal thickness measurements, and to provide a baseline measurement for comparison with later time-points. Additional OCT imaging was undertaken at 6-months post-injection in all groups. Dark- and light-adapted electroretinography was undertaken at 2-, 4- and 6-months post-injection.
In the high-dose group (1.5×108 gc), an extension study was undertaken with electroretinography performed at 8-, 10- and 12-months post-injection with additional OCT imaging performed at 12- and 18-months post-injection and OMR testing at 18 to 22 months post-injection for both Cdhr1−/− and C57BL6J groups (
At 4-weeks post-injection, outer retinal thickness measurements did not differ between paired AAV- and PBS-injected eyes at equivalent superior retinal loci (P=0.99 for 1.5×108 gc and P=0.97 for 1.5×107 gc, two-way ANOVA) on OCT imaging in C57BL6J mice (
Dark-adapted electroretinography did not detect a difference between low dose (1.5×107 gc) AAV- and PBS-injected paired control eyes at 6 months post-injection (
The dark-adapted electroretinography luminance series identified a benefit of sub-retinal AAV8.GRK1.CDHR1.pA as early as 2-months post-injection when A-wave responses were significantly improved compared to PBS-injected control eyes (P<0.0001 across all light intensities; two-way ANOVA;
Raw ERG traces recorded simultaneously from paired Cdhr1−/− eyes 12-months post-injection illustrate the benefit of CDHR1 gene therapy (
Following high-dose AAV8.GRK1.CDHR1.pA, a significant benefit was seen in dark-adapted flicker responses at 2-month post-injection compared to PBS-injected control eyes (P<0.0001; n=28). At this early timepoint, other light-adapted single flash and flicker response amplitudes were better preserved in AAV- versus PBS-injected eyes, although not significant individually (
In the low dose group, although mean response amplitudes were greater in AAV-injected n=23), multiple comparison testing failed to detect a significant benefit in any individual light-adapted or flicker ERG test (
Raw light-adapted ERG traces recorded simultaneously 12-months post-injection in a single Cdhr1−/− mouse illustrate the benefits of high-dose AAV-injection on cone photoreceptor function that is sustained to at least 12-months post-injection (
Dark-adapted A-wave implicit times were significantly shorter in AAV- versus PBS-injected Cdhr1−/− eyes (mean, 6.0 vs. 10.2 ms at 25 cd·s/m2) at 2-months post- injection in the high-dose group (P<0.0001 at all light intensities; n=28), a difference which increased to 12-months post-injection when A-waves were not easily discernible in PBS-injected eyes (
Conversely, A-wave implicit times were significantly longer in Cdhr1−/− versus C57BL/6J eyes (P<0.0001 overall, and at the 4 highest light intensities) at 2-months following PBS injection. In the low dose group, A-wave implicit times were not significantly different between AAV- and PBS-injected eyes to 6-months post-injection in Cdhr1−/− mice (P=0.43; n=22) (
Sub-retinal injection of AAV8.GRK1.CDHR1.pA at a dose of 1.5×108 shortened A-wave implicit times on light-adapted, single flash ERG versus PBS-injected control eyes at 2-months post-injection (P=0.03; n=28) in Cdhr1−/− mice (
In the low dose group, A-wave implicit times on light-adapted single flash ERG were no different in AAV and PBS-injected eyes at 2 months (P=0.73, n=24). However, A-wave implicit times were shorter in AAV-injected eyes at 6 months post-injection overall (P=0.018; n=22) (
In the high-dose group, there was no difference in A-wave amplitudes (P=0.052; n=19) and B-wave amplitudes (P=0.55) in C57BL/6J mice 12-months post- injection, with no statistical difference between amplitudes at any light intensity on multiple comparison testing. The two groups were well-matched at all earlier timepoints (
There was no difference in light-adapted single-flash and flicker response amplitudes between high dose AAV- and PBS-injected eyes to 12-months post injection in C57BL/6J mice (P>0.99 overall; n=19), with no differences in individual ERG tests of the cone system on multiple comparison testing (P>0.97 for all tests) (
A-wave implicit times on light-adapted, single-flash ERG at both 3 and 10 cd·s/m2, were no different between AAV- and PBS-injected eyes in C57BL/6J mice in both low- (P=0.96) and high-dose (P=0.26) groups at 6- and 12-months post-injection, respectively (
Natural history data are presented identifying the extinction of photopic and scotopic OMR in Cdhr1−/− mice at 20-24 months of age, whilst responses remained identifiable in age-matched C57BL/6J controls. Optomotor responses were preserved in AAV-injected Cdhr1−/− eyes (n=20) under photopic conditions (1000 lux) at 19-months, and scotopic conditions (0.01 lux) at 21-months post-injection at two speeds of rotation of the OKN drum; P<0.0001 for AAV-versus PBS-injected eyes for all speeds and at both levels of illuminance (
Optomotor testing under photopic (20-months) and scotopic (22-months post-injection) conditions did not identify a difference between AAV- and PBS-injected eyes in C57BL/6J mice at either 15 s or 30 s per rotation of the OKN drum (
In Cdhr1−/− mice, AAV-injection modified the relationship between structural and functional measures seen in PBS-injected and untreated control eyes, further supporting a slowing of photoreceptor degeneration (R2>0.98 for all groups,
In C57BL/6J mice, high dose AAV8.GRK1.CDHR1.pA injection did not affect the structure-function correlation observed in PBS-injected and untreated control eyes, further supporting other observations of safety of CDHR1 gene therapy.
Functional rescue of rod and cone photoreceptor responses were well matched in AAV-injected eyes relative to age- and treatment-matched C57BL/6J mice (
Retinal thickness measurements undertaken 4-weeks post-injection did not reveal differences between high-dose AAV- (1.5×108 gc) and PBS-injected eyes at equivalent superior retinal loci (
At 1.5×108 gc, inferior retinal locations which were presumed not to be detached as part of the surgical procedure were also significantly thicker in AAV- versus PBS-injected control eyes at 6-months (58.0 μm vs 52.1 μm; P=0.012). This effect remained statistically significant at 12-months although with a smaller effect size (37.5 μm vs 30.3 μm; n=21; P=0.0032) and not significant at 18 months post-injection (P=0.125; n=20). However, retinal structure was preserved in the superior half of the inferior retina (
At the low dose of 1.5×107 gc, retinal thickness measurements at superior retinal locations were not significantly different at 4-weeks post-injection (mean 75.9 μm versus 79.4 μm; P=0.18, two-way ANOVA;
In addition to preservation of the outer retina, as defined as the distance between the outer plexiform layer and retinal pigment epithelium, several morphological changes were evident in Cdhr1−/− mice following high-dose (1.5×108 gc) AAV8.GRK1.CDHR1.pA. As early as 1-month after subretinal AAV injection, 11 of 28 mice showed evidence of lengthening of the photoreceptor outer segment band on OCT imaging (
High-dose AAV.GRK1.CDHR1.pA increased the reflectivity of the ellipsoid zone and preserved the external limiting membrane in Cdhr1−/− mice to 18-months post-injection. In Cdhr1−/− mice, the EZ is no longer discernible on OCT imaging after 6-months of age. Moreover, when present, the EZ appears less reflective compared to age-matched wildtype controls, appearing similar in width and reflectivity to the ELM (
There were no specific morphological alterations seen in C57BL6J mice in either AAV-dose group.
Following high dose AAV8.GRK1.CDHR1.pA injection (1.5×108 vg), preservation and regeneration of the OCT layer representing the photoreceptor OS was identified in AAV-injected eyes to 18-months post-injection on OCT imaging (
High-dose AAV8.GRK1.CDHR1.pA was found to lengthen the layer representing photoreceptor OS on OCT imaging in Cdhr1−/− mice as early as 1-month post-injection, which increased in radial extent across the superior retina to 18-months post-injection (P<0.0001 at all timepoints vs. PBS-injected eyes;
Transmission electron microscopy confirmed OS regeneration following high dose AAV8.GRK1.CDHR1.pA in Cdhr1−/− eyes to 21-months post-injection (
In PBS-injected Cdhr1−/− mice, progressive, patchy RPE atrophy was demonstrated, most evident on NIR-AF imaging at 18-months post-injection (
The present Examples demonstrate the first therapy shown to ameliorate progressive photoreceptor degeneration in a validated model of CDHR1-associated retinal degeneration. In Cdhr1−/− mice, sub-retinal delivery of AAV8.CDHR1 rescued A-wave amplitudes (p<0.0001 at all time points) and B-wave amplitudes (p<0.0001 from 6 months) on dark-adapted electroretinography when compared with PBS-injected control eyes. Light-adapted flicker ERG amplitudes were greater in AAV-treated eyes at 10-months post-injection (p<0.0001). Retinal imaging findings consistent with regeneration of the photoreceptor outer segment were only identified in AAV-treated eyes, with therapeutic effect seen as early as 1-month post-injection (p=0.001). CDHR1 gene therapy reduced the rate of photoreceptor cell death as indicated photoreceptor layer thickness measurements compared to controls at 6-months post-injection (p<0.0001). Sub-retinal delivery of AAV.CDHR1 was safe in C57BL6J mice as evaluated by structural and functional measures. AAV-mediated expression of the human CDHR1 transgene in the Cdhr1−/− murine retina rescued cone and rod photoreceptor function through restoration of photoreceptor outer segment band and slowing of photoreceptor cell death. These beneficial structural effects were mostly limited to the superior retina of AAV-treated eyes, although mild inferior structural preservation and outer segment regeneration was attributable to a reduced level of CDHR1 expression shown in the inferior retina.
AAV8.GRK1.CDHR1.pA delivered by sub-retinal injection at a dose of 1.5×108 vg preserved rod- and cone-photoreceptor response amplitudes and reduced light- and dark-adapted photoreceptor response implicit times on electroretinography to at least 12-months post-injection in Cdhr1−/− mice when compared to PBS-injected control eyes. A slowing of photoreceptor cell death, complete regeneration of rod photoreceptor outer segment length, alignment and morphology on electron microscopy, increased reflectivity of the ellipsoid zone and preservation of the ELM on OCT imaging were seen only in AAV-injected eyes, with the treatment effect sustained to at least 21-months post-injection. A clear benefit of high-dose AAV8.GRK1.CDHR1.pA on visual behaviour was demonstrated on photopic and scotopic optomotor testing, whilst responses were absent in PBS-injected eyes at 19-21 months post-injection—the most durable therapeutic response of any pre-clinical murine study of AAV retinal gene therapy. Longitudinal structural, functional and behavioural testing, using identical experimental protocols, did not detect any significant toxic effects of AAV8.GRK1.CDHR1.pA at 1.5×108 vg in C57BL/6J mice to 22-months post-injection when compared to PBS-injected control eyes, except for very mild thinning on OCT imaging in matched superior retinal locations measured at 18-months post-injection. A dose of 1.5×107 vg was neither significantly therapeutic in Cdhr1−/− mice, nor toxic in C57BL/6J mice to 6-months post-injection.
Hence, a single sub-retinal injection of 1.5×108 vg of AAV.GRK1.CDHR1.pA delivered at 3-4 weeks of age preserved rod- and cone-photoreceptor response amplitudes, reduced rod- and cone photoreceptor response implicit times, slowed photoreceptor cell death, preserved outer retinal structures (e.g. ELM and EZ on OCT imaging) and regenerated full-length, morphologically normal photoreceptor outer segments at 22-months post-injection in AAV-injected Cdhr1−/− eyes. A clear beneficial effect on visual behaviour through photopic and scotopic optomotor testing was demonstrated in AAV-injected eyes at 19-21 months post-injection. The treatment benefit persisted to at least 21-months post-injection, the most durable response following AAV-gene therapy demonstrated in any pre-clinical murine model of retinal degeneration to date.
PBS-injected Cdhr1−/− control eyes exhibited severe outer retinal degeneration without identifiable OS, and with minimal or absent functional responses at the equivalent timepoints. Evidence of a true therapeutic effect was further supported by the dose-dependent and location-dependent nature of the observed therapeutic effects (i.e. using the inferior retina as an internal control) and incremental therapeutic effects on both longitudinal structural and functional analyses, with a greater therapeutic effect size demonstrated across four timepoints on OCT imaging and six timepoints on dark- and light-adapted ERG.
CDHR1 functions in the development of nascent photoreceptor outer segment discs and the higher order organisation of the outer segment. Restoration of the CDHR1 transgene to rod and cone photoreceptors may result in progressive regeneration of photoreceptor outer segments as demonstrated on OCT imaging and confirmed on electron microscopy in this study. Moreover, this beneficial morphological correction appears to begin as early as 1-month, increases to 12-months, and persists to at least 21-months post-injection. Late OS regeneration (i.e. seen at 6-months onwards) may reflect the time required for photoreceptor transduction, a process which has been shown to further increase between 6- and 12-months post-injection in the murine retina. Moreover, the process of outer segment renewal occurs at a rate of approximately 2.3 μm per day with the outer segment measuring approximately ˜20-25 μm in C57BL/6J mice. Cadherins form a superfamily of proteins characterised by presence of two or more extracellular cadherin repeats. Through homophilic or heterophilic interactions, cadherins serve crucial roles in cell-to-cell adhesion. In highly evolved sensory cells, such as the photoreceptors in the retina and the hair cells of the inner ear, cadherins serve more complex functions, principally through heterophilic interactions with other proteins to support the function of the cilia. To our knowledge, this is the first time that a transgene expressing a cadherin has been shown to ameliorate a disease phenotype in vivo.
The observation of outer segment regeneration following AAV8.CDHR1 gene therapy suggests successful heterophilic interaction of CDHR1 with an as yet unidentified binding partner within the periciliary ridge of the inner segment. Moreover, lengthening of the outer segments following gene therapy suggests successful uncoupling of CDHR1-based connections to the inner segment following horizontal outer segment disc elongation. This further suggests successful post-translational modification of the expressed human CDHR1 protein.
A better characterisation of the natural history of CDHR1-related retinal degeneration will help to determine the most appropriate timing, and support the ethical approval for intervention in a given patient. Original data collected involving an international collaboration identified 149 individuals with retinal degeneration due to biallelic CDHR1 variants. Furthermore, the study has established key aspects of the natural history of the disease. All individuals with biallelic null variants in CDHR1, as modelled by the Cdhr1−/− mouse are blind by the age of 60 (
Using a published methodology that uses the Hardy-Weinberg equilibrium to calculate the carrier frequency and genetic prevalence of disease (PMID: 31964843), we calculated the global genetic prevalence of CDHR1-associated retinal degeneration using all validated disease-associated variants (including ˜60 novel variants identified in the international collaborative study. We calculated a genetic prevalence estimate for all six major characterised world populations (European, European Finnish, African, South Asian, East Asian, Latino) based on the allele frequency of all reported pathological CDHR1 variants in that population.
This calculation predicts more than 200,000 individuals worldwide who are expected to manifest or to later develop retinal degeneration due to CDHR1 variants, as shown in Table 2.
In Europe and North America, an estimated 62,665 individuals are expected to be affected (˜1 in 20,902 individuals), with a carrier frequency of 1 in 73.
Furthermore, by establishing the retinal phenotype associated with each variant, we were able to calculate the following genetic prevalence estimates by retinal phenotype, as shown in Tables 3 and 4.
Patients in group 1 are homozygous for c.783G>A variant, patients in group 2 have one c.783G>A variant and one truncating variant, and patients in group 3 have two truncating variants in CDHR1. In retinal imaging using fundus autofluorescence (488 nm), near infra-red autofluorescence (790 nm), SD-OCT central, well-defined area of retinal atrophy can be seen in most patients except for patient 1 who has an early stage of disease. Group 1 patients have qualitatively normal fundus autofluorescence and OCT imaging outside the central area of atrophy. Group 2 patients have some evidence of structural abnormalities outside of the area of macular atrophy. Group 3 patients have significant peripheral retinal degeneration with RPE loss. SD-OCT imaging demonstrates interruption of the EZ in patient 1.1 (early disease) and IZ with eventual loss of these layers and the RPE in the central zone with ONL thinning. Patient 2.2 has generalised thinning of the ONL beyond the central area. Patients in group 3 have generalised outer retinal thinning beyond the central area of macular atrophy.
This series in particular highlights the relatively high frequency of patients with the hypomorphic variant (c.783G>A) in CDHR1. This variant leads to in-frame exon skipping an unusual phenotype with early macular involvement without peripheral degeneration. Patients in this group develop visual symptoms in their 5th decade which progresses to severe loss of central vision within 10 years. However, patients with two null variants in CDHR1 present earlier with more severe visual loss affecting the central and peripheral retina on functional testing as characterised on visual field testing and ERG. Patients with one c.783G>A variant and one null variant have an intermediate phenotype. This data suggests a wide-therapeutic window for intervention with gene replacement during which time the natural history of disease may be ameliorated.
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VDRHSGVLRLQAGATLDYERSRTHYITVVAKDGGGRLHGADVVFSATTTVTVNVEDVQDMAPVFVGTPYYGYVYEDTLPGSEVLKVVAMDG
DRGKPNRILYSLVNGNDGAFEINETSGAISITQSPAQLQREVYELHVQVTEMSPAGSPAAQATVPVTIRIVDLNNHPPTFYGESGPQNRFE
LSMNEHPPQGEILRGLKITVNDSDQGANAKENLQLVGPRGIFRVVPQTVLNEAQVTIIVENSAAIDFEKSKVLTFKLLAVEVNTPEKFSST
ADVVIQLLDTNDNVPKFDSLYYVARIPENAPGGSSVVAVTAVDPDTGPWGEVKYSTYGTGADLFLIHPSTGLIYTQPWASLDAEATARYNF
YVKAEDMEGKYSVAEVFITLLDVNDHPPQFGKSVQKKTMVLGTPVKIEAIDEDAEEPNNLVDYSITHAEPANVFDINSHTGEIWLKNSIRS
LDALHNITPGRDCLWSLEVQAKDRGSPSFSTTALLKIDITDAETLSRSPMAAFLIQTKDNPMKAVGVLAGTMATVVAITVLISTATFWRNK
| Number | Date | Country | Kind |
|---|---|---|---|
| 2104611.5 | Mar 2021 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2022/050800 | 3/30/2022 | WO |
