Patent Application
20230064326
The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is VEDE_009_03US_SeqList_ST26. The text file is about 168 KB, created on Jul. 11, 2022, and is being submitted electronically via Patent Center.
The present invention is directed to the fields of gene therapy, retinal disease and vision restoration.
There is an unmet need for effective treatments for vision loss that address underlying condition through targeted gene therapy in particular cell types of interest. The present disclosure meets this need and offers other related advantages.
In one aspect of the present disclosure, there are provided nucleic acid vectors comprising a CBh promoter sequence operably linked to a heterologous sequence encoding a G-protein coupled receptor (GPCR). In some embodiments, the CBh promoter comprises: (i) a cytomegalovirus (CMV) enhancer sequence and (ii) a chicken beta actin (CBA) promoter sequence. In some embodiments, the CBh promoter comprises: (i) a cytomegalovirus (CMV) enhancer sequence, (ii) a chicken beta actin (CBA) promoter sequence, and (iii) an intron sequence. In more particular embodiments, the CBh promoter comprises: (i) a cytomegalovirus (CMV) enhancer sequence, (ii) a chicken beta actin (CBA) promoter sequence, and (iii) a hybrid intron sequence comprising a CBA intron sequence and a Mirabilis mosaic virus (MMV) intron sequence.
In some embodiments of the nucleic acid vectors of the disclosure, the CBh promoter comprises the sequence of SEQ ID NO: 1 or a functional fragment or variant thereof having at least 90% identity thereto, where the functional fragment or variant is capable of directing expression of the heterologous sequence in the retina. In more particular embodiments, the CBh promoter comprises the sequence of SEQ ID NO: 1.
In some embodiments of the nucleic acid vectors of the disclosure, the heterologous sequence further comprises a sequence encoding an affinity tag in addition to the GPCR. In more particular embodiments, the affinity tag comprises a SNAP polypeptide, or a functional fragment or variant thereof. In more particular embodiments, the SNAP polypeptide comprises the sequence of SEQ ID NO: 47 or SEQ ID NO: 48 or a functional fragment or variant thereof having at least 90% identity thereto. In more particular embodiments, the SNAP polypeptide is a polypeptide that binds benzylguanine (and/or to a photoswitch conjugate comprising benzylguanine).
In some embodiments of the nucleic acid vectors of the disclosure, the GPCR is an inhibitory G-protein (Gi)-coupled GPCR. In some embodiments, the GPCR is a stimulatory G-protein (Gq)-coupled GPCR. In some embodiments, the GPCR is a stimulatory G-protein (Gs)-coupled GPCR. In some embodiments, the GPCR comprises a metabotropic glutamate receptor (mGluR). In more specific embodiments of the invention, the GPCR sequence comprises a functional fragment or variant of a GPCR sequence. In other more specific embodiments, the functional fragment or variant thereof retains one or more desired activities of a wild type GPCR, and has at least 70%, at least 80%, at least 90%, at least 95% or at least 99% or more identity the sequence of a wild type human GPCR.
In some embodiments of the nucleic acid vectors of the disclosure, the heterologous sequence encodes a fusion protein comprising the affinity tag and the GPCR, such as wherein the fusion protein comprises, from amino (N) to carboxy (C) ends, the SNAP sequence and the GPCR sequence.
In some embodiments, the heterologous sequence may further comprise or encode additional sequences, such as signal peptides, linkers and the like. For example, in some embodiments, the heterologous sequence encodes a fusion protein, which, in addition to comprising the affinity tag and the GPCR, also comprises a signal peptide (SP) at its N-terminus. Thus, a fusion protein of the disclosure can also comprise, from amino (N) to carboxy (C) ends, a signal peptide sequence, an affinity tag sequence (e.g., a SNAP sequence) and a GPCR sequence, optionally with linker sequences between one or more of these elements. In some embodiments, the signal peptide is cleaved and is not part of the final functional protein expressed in vivo. However, in some cases, its presence is needed to facilitate proper trafficking to the membrane and/or to serve other purposes. The signal peptide may be native to the GPCR being expressed or may correspond or be derived from the signal peptide of another GPCR protein sequence.
In certain embodiments of the nucleic acid vectors of the disclosure, the GPCR used in accordance with the present invention is an mGluR polypeptide. In other more particular embodiments, the sequence encoding the mGluR polypeptide comprises one or more of: (a) a nucleic acid sequence isolated or derived from a human mGluR sequence; (b) a nucleic acid sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% or more identity the sequence of (a); and (c) a codon-optimized sequence derived from the sequence of any one of (a)-(c).
In some embodiments of the nucleic acid vectors of the disclosure, the mGluR comprises one or more of: (a) an amino acid sequence isolated or derived from a human mGluR sequence; (b) an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% or more identity to a human mGluR sequence of (a); (c) an amino acid sequence having one or more variations conserved between a human mGluR sequence and at least one non-human mammal; and (d) an amino acid sequence having one or more silent mutations when compared to the sequence of any one of (a)-(c).
In some embodiments of the nucleic acid vectors of the disclosure, the mGluR comprises one or more of mGluR1, mGluR2, mGluR3, mGluR4, mGluR5, mGluR6, mGluR7, and mGluR8, or a functional fragment or variant thereof. In other more specific embodiments, the functional fragment or variant thereof retains one or more desired activities of a wild type mGluR, and has at least 70%, at least 80%, at least 90%, at least 95% or at least 99% or more identity the sequence of a wild type human mGluR.
In some embodiments, the mGluR comprises mGluR2. In some embodiments, the sequence encoding an mGluR comprises a sequence encoding a human mGluR2.
In some embodiments of the nucleic acid vectors of the disclosure, the sequence encoding a human mGluR2 comprises the nucleic acid sequence of SEQ ID NO: 8, or a functional fragment or variant thereof.
In some embodiments of the nucleic acid vectors of the disclosure, the human mGluR2 comprises the amino acid sequence of SEQ ID NO: 9, or a functional fragment or variant thereof.
In some embodiments of the nucleic acid vectors of the disclosure, the vector further comprises one or more of a sequence comprising an enhancer, a sequence comprising an intron or any portion thereof, a sequence comprising an exon or any portion thereof, a sequence comprising a Kozak sequence, a sequence comprising a post-transcriptional response element (PRE), a sequence comprising an inverted terminal repeat (ITR) sequence, a sequence comprising a long terminal repeat (LTR) sequence, and a poly-A sequence.
In some embodiments of the nucleic acid vectors of the disclosure, the vector further comprises a linking element in between one or more of the elements that make up the vector. For example, in some embodiments, the vectors of the disclosure comprise a linker sequence which is located between the signal peptide and the start of the affinity tag (e.g., a SNAP tag sequence). In some embodiments, the linker sequence may be part of the final functional protein but in other cases it may not. In a specific embodiment, a linker sequence comprising the amino acid sequence TRTRGS is located between the signal peptide and the start of the affinity tag sequence.
In some embodiments of the nucleic acid vectors of the disclosure, the vector further comprises a cleaving element. In more specific embodiments, the cleaving element comprises as self-cleaving element.
In some embodiments of the nucleic acid vectors of the disclosure, the vector further comprises a multicistronic element. In more specific embodiments, the multicistronic element comprises an IRES sequence.
According to another aspect of the present disclosure, there are provided delivery vectors and systems for delivering a nucleic acid vector of the disclosure comprising a CBh promoter operably linked to a sequence encoding a GPCR, such as an mGluR, to the cell type of interest. In some embodiments, the delivery vector is a viral vector. In some embodiments, the viral vector is an adeno-associated vector (AAV). In some embodiments, the AAV is a recombinant AAV (rAAV). In some embodiments, the rAAV comprises a sequence isolated or derived from an AAV of a first serotype and a sequence isolated or derived from an AAV of a second serotype. In some embodiments, the rAAV comprises a capsid sequence isolated or derived from an AAV of a serotype and a heterologous capsid insert sequence. In some embodiments, the heterologous capsid insert sequence is neither isolated nor derived from an AAV of any known serotype. In some embodiments, the heterologous capsid insert sequence comprises a random sequence.
In some embodiments of the disclosure, the delivery vector targets a retinal cell type. In some embodiments, the retinal cell type is a neuron. In some embodiments, the retinal cell type is a retinal ganglion cell, a horizontal cell, an amacrine cell, a bipolar cell or a photoreceptor cell. In some embodiments, the retinal cell type is not a photoreceptor. In some embodiments, the retinal cell type is not a retinal ganglion cell. In some embodiments, the retinal cell type is not a horizontal cell. In some embodiments, the retinal cell type is not an amacrine cell. In some embodiments, the retinal cell type is not a bipolar cell. In some embodiments, the delivery vector targets a Muller cell or an astrocyte.
According to another aspect of the present disclosure, there are provided cells, such as human cells, which have been genetically modified to contain a nucleic acid vector of the disclosure, such as a vector comprising a CBh promoter operably linked to a sequence encoding a GPCR.
According to another aspect of the present disclosure, there are provided pharmaceutical compositions comprising a nucleic acid vector, delivery vector and/or cells of the disclosure, in combination with a pharmaceutically acceptable carrier.
According to another aspect of the present disclosure, there are provided methods of treating a disease or disorder, comprising administering to a subject in need thereof, a therapeutically effective amount of a nucleic acid vector of the disclosure, an expression vector of the disclosure, a delivery vector of the disclosure, a cell of the disclosure or a pharmaceutical composition of the disclosure.
In some embodiments, the disease or disorder to be treated comprises a retinal disease or disorder. In some embodiments, the retinal disease or disorder comprises a decrease or an inhibition of a function of one or more retinal neurons. In some embodiments, the one or more retinal neurons comprise a photoreceptor cell, a cone cell, a rod cell, a ganglion cell, a bipolar cell, an amacrine cell, and a horizontal cell. In some embodiments, the one or more retinal neurons does not comprise a rod cell or a cone cell. In some embodiments, the one or more retinal neurons does not comprise a ganglion cell. In some embodiments, the one or more retinal neurons does not comprise a bipolar cell. In some embodiments, the one or more retinal neurons does not comprise an amacrine cell. In some embodiments, the one or more retinal neurons does not comprise a horizontal cell.
In some embodiments of the treatment methods of the disclosure, the subject has experienced or is at risk of experiencing a loss of visual acuity. In some embodiments, the subject has acquired condition resulting in decreased visual acuity when compared to an individual lacking the acquired condition. In some embodiments, the acquired condition comprises one or more of trauma, injury, degeneration, infection, decreased function of one or more retinal proteins, decreased activity of one or more retinal proteins, decreased expression of one or more retinal transcripts (RNA or DNA), decreased translation of one or more retinal transcripts (RNA or DNA), increased turnover of one or more retinal proteins or retinal transcripts resulting in decreased expression of one or more retinal proteins, decreased intracellular signaling of one or more retinal cell types (optionally, in response to a signal from another cell or from the environment such as light), and/or decreased intercellular signaling between retinal cells or between retinal structures (optionally, in response to a signal from another cell or from the environment such as light). In some embodiments, the subject has a congenital condition resulting in decreased visual acuity when compared to an individual lacking the congenital condition. In some embodiments, the congenital condition comprises color blindness.
In some embodiments of the methods of the disclosure, the retinal disease or disorder comprises degeneration of one or more retinal neurons or degeneration of a function of one or more retinal neurons. In some embodiments, the retinal disease or disorder comprises loss of cell viability or cell death of one or more retinal neurons.
In some embodiments of the methods of the disclosure, the administering comprises an intraocular route. In some embodiments, the intraocular route comprises an intravitreal or a subretinal route. In some embodiments, the administering comprises an injection, infusion, engraftment or implantation.
In some embodiments of the methods of the disclosure, a therapeutically effective amount of a composition of the disclosure restores or enhances visual acuity compared to a reference level of visual acuity. In some embodiments, the reference level of visual acuity comprises a medically accepted standard for an age-matched healthy individual. In some embodiments, the reference level of visual acuity comprises a baseline level of the subject measured either prior to disease onset or prior to treatment. In some embodiments, the reference level of visual acuity comprises a level of visual acuity measured in an unaffected or untreated eye of the subject.
As described herein, the present disclosure relates generally to optogenetic compositions and methods for use. Exemplary compositions according to the disclosure include nucleic acid vectors comprising a CBh promoter sequence operably linked to a heterologous sequence encoding a G-protein coupled receptor (GPCR), as well as the use of such vectors in the therapeutic treatment of ocular diseases and disorders.
The following definitions and descriptions are provided to better understand the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
A “promoter” is generally understood as a nucleic acid sequence that is recognized by an RNA polymerase which binds to the promoter and directs transcription of a nucleic acid sequence operably linked to the promoter. A promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter can also optionally include enhancer or repressor elements. An inducible promoter is generally understood as a promoter that mediates transcription of an operably linked gene in response to a particular stimulus.
The term “enhancer” refers to a nucleic acid sequence which contains sequences capable of providing enhanced transcription and in some instances can function independent of their orientation relative to another control sequence. An enhancer can function cooperatively or additively with promoters and/or other enhancer elements.
The terms “heterologous gene” or “heterologous nucleic acid” or “heterologous sequence”, as used herein, refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous nucleic acid in a host cell can include sequences that are endogenous to the particular host cell but where the sequences have been modified from their wild type forms. A heterologous sequence can also include a sequence that is endogenous to the particular host cell but is under the control of a promoter sequence that is not naturally associated with the sequence. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence.
“Operably-linked” or “functionally linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other in an intended manner. For example, a regulatory DNA sequence (such as a promoter) is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation. The two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell. In some embodiments, a sequence encoding a CBh promoter is operably linked to a sequence encoding a GPCR receptor, which may or may not be contiguous sequences, but are operably linked because the promoter is capable of driving expression of the GPCR receptor in a cell.
The term “vector” is used herein to refer to a nucleic acid molecule capable transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule. A vector may include sequences that direct autonomous replication in a cell or may include sequences sufficient to allow integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, e.g., replication defective retroviruses and lentiviruses.
A “transcribable nucleic acid molecule” as used herein refers to any nucleic acid molecule capable of being transcribed into a RNA molecule.
The “transcription start site” or “initiation site” is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site all other sequences of the gene and its controlling regions can be numbered. Downstream sequences (i.e., further protein encoding sequences in the 3′ direction) can be denominated positive, while upstream sequences (mostly of the controlling regions in the 5′ direction) are denominated negative.
A “construct” is generally understood as any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecule has been operably linked.
A construct of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3′ transcription termination nucleic acid molecule. In addition, constructs can include but are not limited to additional regulatory nucleic acid molecules from, e.g., the 3′-untranslated region (3′ UTR). Constructs can include but are not limited to the 5′ untranslated regions (5′ UTR) of an mRNA nucleic acid molecule which can play an important role in translation initiation and can also be a genetic component in an expression construct. These additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.
Methods are known for introducing constructs into a cell in such a manner that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product.
Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit translation of a specific RNA molecule of interest. For the practice of the present disclosure, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754).
The term “transformation” refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as “transgenic” cells, and organisms comprising transgenic cells are referred to as “transgenic organisms”.
“Transformed,” “transgenic,” and “recombinant” refer to a host cell or organism such as a bacterium, cyanobacterium, animal or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome as generally known in the art and disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. The term “untransformed” refers to normal cells that have not been through the transformation process.
As used herein, the term “genetically engineered” or “genetically modified” refers to the addition of extra genetic material in the form of DNA or RNA into the total genetic material in a cell. The terms, “genetically modified cells”, “modified cells”, and “redirected cells” are used interchangeably. As used herein, the term “gene therapy” refers to the introduction of extra genetic material in the form of DNA or RNA into the total genetic material in a cell that restores, corrects, or modifies expression of a gene, or for the purpose of expressing a therapeutic polypeptide.
“Wild-type” refers to a virus or organism found in nature without any known mutation.
Design, generation, and testing of the variant nucleotides, within transcriptional regulatory sequences (e.g., promoters) as well as encoded polypeptides, having the herein required percent identities and retaining a required promoter activity or activity of the expressed protein is within the skill of the art. For example, directed evolution and rapid isolation of mutants can be according to methods described in references including, but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688; Sanger et al. (1991) Gene 97(1), 1 19-123; Ghadessy et al. (2001) Proc Natl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art could generate a large number of nucleotide and/or polypeptide variants having, for example, at least 90-99% identity or 95-99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art.
In some embodiment, a CBh promoter variant sequences comprises one or more nucleotide insertions, deletions, substitutions, or modifications, relative to the specific CBh promoter sequences disclosed herein, such that increased or stabilized CBh promoter activity is achieved. In some embodiments, a CBh promoter sequence comprises 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 20 or more, or 25 or more, nucleotide insertions, deletions, substitutions, or modifications, relative to the specific CBh promoter sequences disclosed herein, such that increased or stabilized CBh promoter activity is achieved.
Nucleotide and/or amino acid sequence identity percent (%) is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. When sequences are aligned, the percent sequence identity of a given sequence A to, with, or against a given sequence B (which can alternatively be phrased as a given sequence A that has or comprises a certain percent sequence identity to, with, or against a given sequence B) can be calculated as: percent sequence identity=X/Y100, where X is the number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.
Host cells can be transformed using a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, receptor-mediated uptake, cell fusion, electroporation, and the like. The transfected cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.
Exemplary nucleic acids which may be introduced to a vector or host cell include, for example, exogenous sequences or sequences which originate with or are present in the same species, but which are incorporated into recipient cells by genetic engineering methods. The term “exogenous” refers to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express. Thus, the term “exogenous” gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in the exogenous DNA can include DNA which is already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.
In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.
The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended.
For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.
Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
As used throughout the disclosure, the term “isolated” is meant to describe a sequence that is removed from its biological context but is otherwise unchanged in sequence.
As used throughout the disclosure, the term “derived” is meant to describe a sequence that has been modified from a naturally occurring sequence but retains sufficient sequence homology or identity to be recognized as preserving one or more structure-function relationships. In some embodiments, a sequence derived from a human sequence contains one or more modified or synthetic nucleic acids that do not occur in nature but may increase stability or reduce immunogenicity. In some embodiments, a sequence derived from a human sequence contains one or more silent mutations that improve manufacturability while retaining function. In some embodiments, a sequence derived from a human sequence is a recombinant sequence. In some embodiments, a sequence derived from a human sequence is a chimeric sequence.
A CBh promoter sequence of the disclosure typically comprises: (i) a cytomegalovirus (CMV) enhancer sequence, (ii) a chicken beta actin (CBA) promoter sequence and (iii) a hybrid intron sequence comprising a CBA intron sequence and a Mirabilis mosaic virus (MMV) intron sequence. In some embodiments, a CBh promoter sequence comprises a sequence as set out in Grey et al. (Hum Gene Therapy 22(9): 1143-1153, 2011). In some embodiments of the present disclosure, the CBh promoter comprises or consists essentially of the nucleic acid sequence of SEQ ID NO: 1.
In some embodiments of the disclosure, a hybrid CBh promoter used according to the present disclosure comprises a nucleic acid sequence derived from a CBh promoter as set forth in SEQ ID NO: 1.
In some embodiments, the CMV enhancer sequence of a hybrid CBh promoter of the present disclosure comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or any percentage identity in between to residues 1-305 of SEQ ID NO: 1, or any functional fragment thereof.
In some embodiments, the CBA promoter sequence of a hybrid CBh promoter of the present disclosure comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or any percentage identity in between to residues 306-583 of SEQ ID NO: 1, or any functional fragment thereof.
In some embodiments, the intronic sequence of a hybrid CBh promoter of the present disclosure comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or any percentage identity in between to residues 584-812 of SEQ ID NO: 1, or any functional fragment thereof.
In some embodiments, the sequence encoding a CBh promoter comprises or consists essentially of SEQ ID NO: 1, or any functional fragment thereof capable of directing expression of a heterologous sequence in the retina. A functional fragment may be essentially any length, including sequences comprising at least at least or no more than 100, at least or no more than 200, at least or no more than 300, at least or no more than 400, at least or no more than 500, at least or no more than 600, or at least or no more than 700 or more nucleic acid residues.
In some embodiments, the CBh promoter comprises or consists of a variant nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or any percentage identity in between to a CBh promoter of SEQ ID NO: 1, or any functional fragment thereof effective for directing expression of a heterologous sequence in the retina.
In some embodiments of the disclosure, the heterologous sequence under the control of the CBh promoter further comprises, in addition to a sequence encoding a GPCR, a sequence encoding an affinity tag. In some embodiments, the affinity tag comprises a SNAP polypeptide. In some embodiments, the SNAP polypeptide comprises the sequence of SEQ ID NO: 47 or SEQ ID NO: 48 below.
In some embodiments, there is no methionine in the first position of the SNAP tag sequence because the start methionine is instead at the N-terminus of a signal peptide that is expressed in fusion with the SNAP tag, optionally with a linker sequence between the two.
In some embodiments, the CBh promoter sequence is operably linked to the sequence encoding the GPCR and to the sequence encoding the affinity tag. As such, the heterologous sequence encodes a fusion polypeptide comprising an affinity tag (e.g., SNAP) and a GPCR.
In some embodiments, the SNAP polypeptide is a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or more amino acid sequence identity to the SNAP sequence set out herein. The SNAP polypeptides and variants used according to the present disclosure are generally those that retain binding to a molecule comprising benzylguanine.
In some embodiments, the nucleic acid vectors of the disclosure are used in conjunction with a photoisomerizable small molecule. In some embodiments, the heterologous sequence comprises a sequence encoding an affinity tag and the photoisomerizable small molecule is capable of binding to the affinity tag to generate an activated affinity tag. In some embodiments, the photoisomerizable small molecule is capable of binding to the affinity tag covalently. In some embodiments, the photoisomerizable small molecule is capable of binding to the affinity tag non-covalently. In some embodiments, the activated affinity tag is capable of binding to the GPCR to produce an activated GPCR. In some embodiments, a SNAP polypeptide of the disclosure binds to a benzylguanine molecule that is associated with a photoisomerizable small molecule. In some embodiments, the photoisomerizable small molecule comprises azobenzene.
In certain more specific embodiments of the present invention, a composition of the present invention, comprising a CBh promoter sequence and a heterologous sequence encoding a GPCR or encoding a fusion polypeptide such as a SNAP-GPCR fusion polypeptide, may be made and used in conjunction with photoisomerizable small molecules in accordance with the disclosures set forth in WO2019/060785 and/or WO2021/243086, the contents of which are incorporated herein by reference in their entireties.
Metabotropic glutamate receptors (mGluRs) of the disclosure may be isolated or derived from any species. In some embodiments, the mGluR comprises one or more of mGluR1, mGluR2, mGluR3, mGluR4, mGluR5, mGluR6, mGluR7 and mGluR8, or a functional fragment or variant thereof.
In some embodiments, the sequence encoding a human mGluR1 comprises or consists of the nucleic acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q13255-1; GenBank Accession No. NM_001278064.2 and SEQ ID NO: 2):
In some embodiments, the human mGluR1 comprises or consists of the amino acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q13255-1, isoform 1; and SEQ ID NO: 3):
In some embodiments, the sequence encoding a human mGluR1 comprises or consists of the nucleic acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q13255-2; GenBank Accession No. NM_001278065.2 and SEQ ID NO: 4):
In some embodiments, the human mGluR1 comprises or consists of the amino acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q13255-2, isoform 2; and SEQ ID NO: 5):
In some embodiments, the sequence encoding a human mGluR1 comprises or consists of the nucleic acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q13255-3; GenBank Accession No. NM_001278067.1 and SEQ ID NO: 6):
In some embodiments, the human mGluR1 comprises or consists of the amino acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q13255-3, isoform 3; and SEQ ID NO: 7):
In some embodiments, the mGluR comprises mGluR2. In some embodiments, the sequence encoding an mGluR comprises a sequence encoding a human mGluR2.
In some embodiments, the sequence encoding a human mGluR2 comprises or consists of the nucleic acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q14416; GenBank Accession No. NM_000839.5 and SEQ ID NO: 8):
In some embodiments, the human mGluR2 comprises or consists of the amino acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q14416; GenBank Accession No. NP_000830.2 and SEQ ID NO: 9):
In some embodiments, the signal peptide of mGluR2 is replaced with a signal peptide from another glutamate receptor, such as mGluR5. In some embodiments, the signal peptide from mGluR2 is replaced with the signal peptide from another GPCR, such as mGluR5. For example, in a specific embodiment, a vector of the present disclosure encodes a fusion polypeptide comprising, from N-terminus to C-terminus, a signal peptide derived from mGluR5 (replacing the mGluR2 signal peptide sequence), a linker sequence, a SNAP tag sequence and an mGluR2 sequence. In a more specific embodiment, the fusion polypeptide the following amino acid sequence:
In even more particular embodiments, a heterologous polypeptide sequence encoded within a vector of the disclosure which is driven by a CBh promoter comprises a mGluR5 signal peptide sequence shown below in bold and/or a linker sequence shown below as underlined text and/or a SNAP tag sequence shown below in italics and/or a mGluR2 sequence shown below in normal text, or a functional fragment or variant of any of the foregoing. In a more specific embodiment, the heterologous polypeptide sequence encoded within a vector of the disclosure comprises the sequence set out below as SEQ ID NO: 49.
MVLLLILSVLLLKEDVRGSAQS
TRTRGS
DKDCEMKRTTLDSPLGKLELS
GCEQGLHEIKLLGKGTSAADAVEVPAPAAVLGGPEPLMQATAWLNAYFH
QPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKWKFGEVISYQQLAALA
GNPAATAAVKTALSGNPVPILIPCHRWSSSGAVGGYEGGLAVKEWLLAH
EGHRLGKPGLGTRKKVLTLEGDLVLGGLFPVHQKGGPAEDCGPVNEHRG
In some embodiments, the mGluR comprises mGluR3. In some embodiments, the sequence encoding an mGluR comprises a sequence encoding a human mGluR3.
In some embodiments, the sequence encoding a human mGluR3 comprises or consists of the nucleic acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q14832-1; GenBank Accession No. NM_000840.2 and SEQ ID NO: 10):
In some embodiments, the human mGluR3 comprises or consists of the amino acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q14832-1; GenBank Accession No. NP_000831.2 and SEQ ID NO: 11):
In some embodiments, the sequence encoding a human mGluR3 comprises or consists of the nucleic acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q14832-2; GenBank Accession No. NM_001363522.2 and SEQ ID NO: 12):
In some embodiments, the human mGluR3 comprises or consists of the amino acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q14832-2; GenBank Accession No. NP_001350451.1 and SEQ ID NO: 13):
In some embodiments, the mGluR comprises mGluR4. In some embodiments, the sequence encoding an mGluR comprises a sequence encoding a human mGluR4.
In some embodiments, the sequence encoding a human mGluR4 comprises or consists of the nucleic acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q14833-1; GenBank Accession No. NM_000841.4 and SEQ ID NO: 14):
In some embodiments, the human mGluR4 comprises or consists of the amino acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q14833-1 and SEQ ID NO: 15):
In some embodiments, the human mGluR4 comprises or consists of the amino acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q14833-2 and SEQ ID NO: 17):
In some embodiments, the sequence encoding a human mGluR4 comprises or consists of the nucleic acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q14833-3; GenBank Accession No. NM_001256812.2 and SEQ ID NO: 18):
In some embodiments, the human mGluR4 comprises or consists of the amino acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q14833-3 and SEO ID NO: 19):
In some embodiments, the sequence encoding a human mGluR4 comprises or consists of the nucleic acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q14833-4; GenBank Accession No. NM_001256813.3 and SEQ ID NO: 20):
In some embodiments, the human mGluR4 comprises or consists of the amino acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q14833-4 and SEQ ID NO: 21):
In some embodiments, the sequence encoding a human mGluR4 comprises or consists of the nucleic acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q14833-5; GenBank Accession No. NM_001256809.3 and SEQ ID NO: 22):
In some embodiments, the human mGluR4 comprises or consists of the amino acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q14833-5 and SEQ ID NO: 23):
In some embodiments, the mGluR comprises mGLuR5. In some embodiments, the sequence encoding an mGluR comprises a sequence encoding a human mGluR5.
In some embodiments, the sequence encoding a human mGluR5 comprises or consists of the nucleic acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB P41594-1; GenBank Accession No. NM_001143831.3 and SEQ ID NO: 24):
In some embodiments, the human mGluR5 comprises or consists of the amino acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB P41594-1 and SEQ ID NO: 25):
In some embodiments, the signal peptide corresponding to residues from about 1-22 of SEQ ID NO: 25 above is used in place of the signal peptide of another glutamate receptor, such as mGluR2.
In some embodiments, the sequence encoding a human mGluR5 comprises or consists of the nucleic acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB P41594-2; GenBank Accession No. NM_001384268.1 and SEQ ID NO: 26):
In some embodiments, the human mGluR5 comprises or consists of the amino acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB P41594-2 and SEQ ID NO: 27):
In some embodiments, the human mGluR5 comprises or consists of the amino acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB P41594-3 and SEQ ID NO: 29):
In some embodiments, the mGluR comprises mGluR6. In some embodiments, the sequence encoding an mGluR comprises a sequence encoding a human mGluR6.
In some embodiments, the sequence encoding a human mGluR6 comprises or consists of the nucleic acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB O15303-1; GenBank Accession No. NM_000843.3 and SEQ ID NO: 30):
In some embodiments, the human mGluR6 comprises or consists of the amino acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB O15303-1; GenBank Accession No. NP_000834.2 and SEQ ID NO: 31):
In some embodiments, the mGluR comprises mGluR7. In some embodiments, the sequence encoding an mGluR comprises a sequence encoding a human mGluR7.
In some embodiments, the sequence encoding a human mGluR7 comprises or consists of the nucleic acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q14831-1; GenBank Accession No. NM_000844.4 and SEQ ID NO: 32):
In some embodiments, the human mGluR7 comprises or consists of the amino acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q14831-1 and SEO ID NO: 33):
In some embodiments, the sequence encoding a human mGluR7 comprises or consists of the nucleic acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q14831-2; GenBank Accession No. NM_181874.3 and SEQ ID NO: 34):
In some embodiments, the human mGluR7 comprises or consists of the amino acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q14831-2 and SEQ ID NO: 35):
In some embodiments, the human mGluR7 comprises or consists of the amino acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q14831-3; SEQ ID NO: 37):
In some embodiments, the human mGluR7 comprises or consists of the amino acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q14831-4 and SEQ ID NO: 39):
In some embodiments, the human mGluR7 comprises or consists of the amino acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB Q14831-5; and SEQ ID NO: 41):
In some embodiments, the mGluR comprises mGluR8. In some embodiments, the sequence encoding an mGluR comprises a sequence encoding a human mGluR8.
In some embodiments, the sequence encoding a human mGluR8 comprises or consists of the nucleic acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB O00222-1; GenBank Accession No. NM_001371084.1 and SEQ ID NO: 42):
In some embodiments, the human mGluR8 comprises or consists of the amino acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB O00222-1 and SEQ ID NO: 43):
In some embodiments, the sequence encoding a human mGluR8 comprises or consists of the nucleic acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB O00222-2; GenBank Accession No. NM_001371085.1 and SEQ ID NO: 44):
In some embodiments, the human mGluR8 comprises or consists of the amino acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB O00222-2 and SEQ ID NO: 45):
In some embodiments, the human mGluR8 comprises or consists of the amino acid sequence of the following sequence, or a functional fragment or variant thereof (UniProtKB O00222-3 and SEQ ID NO: 46):
In some embodiments, the sequence encoding the mGluR comprises one or more of: (a) a nucleic acid sequence isolated or derived from a human mGluR sequence; (b) a nucleic acid sequence having at least 70% identity to a human mGluR sequence; (c) a nucleic acid sequence having at least 70% identity to the sequence of (a); and (d) a codon-optimized sequence derived from the sequence of any one of (a)-(c).
In some embodiments of the compositions of the disclosure, the mGluR comprises one or more of: (a) an amino acid sequence isolated or derived from a human mGluR sequence; (b) an amino acid sequence having at least 70% identity to a human mGluR sequence; (c) an amino acid sequence having at least 70% identity to the amino acid sequence of (a); (d) an amino acid sequence having one or more variations conserved between a human mGluR sequence and at least one non-human mammal; and (e) an amino acid sequence having one or more silent mutations when compared to the sequence of any one of (a)-(c).
In some embodiments, the mGluR comprises one or more of mGluR1, mGluR2, mGluR3, mGluR4, mGluR5, mGluR6, mGluR7, and mGluR8. In some embodiments, the mGluR comprises mGluR2. In some embodiments, the sequence encoding an mGluR comprises a sequence encoding a human mGluR2.
In some embodiments, the mGluR comprises or consists of a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or any percentage identity in between to a human mGluR. In some embodiments, the human mGluR comprises or consists of the sequence of one or more of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 18, 20, 22, 24, 26, 30, 32, 34, 42, and 44.
In some embodiments, the mGluR comprises or consists of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or any percentage identity in between to a human mGluR. In some embodiments, the human mGluR comprises or consists of the sequence of one or more of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, and 47.
In some embodiments, the nucleic acid vector may further comprise one or more of a sequence comprising an enhancer, a sequence comprising an intron or any portion thereof, a sequence comprising an exon or any portion thereof, a sequence comprising a Kozak sequence, a sequence comprising a post-transcriptional response element (PRE), a sequence comprising an inverted terminal repeat (ITR) sequence, a sequence comprising a long terminal repeat (LTR) sequence, and a poly-A sequence.
In some embodiments, the nucleic acid vector further comprises a linking element that links one or more elements that are present in a vector of the disclosure and/or a polypeptide encoded by a vector of the disclosure. A linking element of the disclosure may link the sequence encoding the promoter to the sequence encoding the mGluR. Alternatively, or in addition, a linking element of the disclosure may link, reversible or irreversibly the composition to one or more of a surface, a tag, a label (detectable or sequence barcode), a ligand, an epitope, a capture probe, a selectable marker, or a delivery vehicle of the disclosure.
In some embodiments, the nucleic acid vector further comprises a cleaving element. In some embodiments, the cleaving element comprises as self-cleaving element. A cleaving element of the disclosure may be positioned between the sequence encoding the promoter to the sequence encoding the mGluR. Alternatively, or in addition, a cleaving element of the disclosure may be positioned further 5′ or 3′ to the sequence comprising the promoter and the mGluR. In some embodiments, the cleaving element may link, reversible or irreversibly, two or more sequences of the composition. In some embodiments, the cleaving element may link, reversible or irreversibly the composition to one or more of a surface, a tag, a label (detectable or sequence barcode), a ligand, an epitope, a capture probe, a selectable marker, or a delivery vehicle of the disclosure. In some embodiments, the cleaving element may link, reversible or irreversibly, two or more sequences of the composition. In some embodiments, the cleaving element may de-link or un-link one or more of a surface, a tag, a label (detectable or sequence barcode), a ligand, an epitope, a capture probe, a selectable marker, or a delivery vehicle of the disclosure by cleavage of the element. In some embodiments, the cleaving element may de-link or un-link two or more sequences of the composition. In some embodiments, the cleavable element comprises a nucleic acid sequence and the nucleic acid sequence may encode a multicistronic element. In some embodiments, the cleavable element comprises a self-cleaving element. In some embodiments, the cleavable element comprises a sequence encoding a self-cleaving peptide.
In some embodiments, the nucleic acid vector further comprises a multicistronic element. In some embodiments, the multicistronic element comprises an IRES sequence.
The disclosure also provides expression and delivery vectors comprising the nucleic acid vectors described herein. Expression vectors include, but are not limited to, any vector suitable for in vitro or ex vivo delivery of a composition of the disclosure to a cell of the disclosure, by any means. In some embodiments, an expression vector comprises a plasmid. In some embodiments, the plasmid is electroporated into a cell of the disclosure. Expression vectors of the disclosure may also comprise delivery vectors of the disclosure when used to introduce a composition in vitro or ex vivo.
Delivery vectors include, but are not limited to, any vector suitable for in vivo delivery of a composition of the disclosure to a cell of the disclosure when in vivo or in situ (in the context of an intact eye). Delivery vectors of the disclosure include, but are not limited, to viral vectors and non-viral vectors. Exemplary viral vectors include, but are not limited to, adeno-associated vectors of any serotype. Exemplary non-viral vectors include, but are not limited to, lipid vectors, polymer vectors and particle vectors. Lipid vectors include, but are not limited to, liposomes, lipid nanoparticles, micelles, lipid polymersomes, and exosomes. Polymer vectors include, but are not limited to, polymersomes, lipid nanoparticles, and nanoparticles. Particle vectors include, but are not limited to, nanoparticles of all geometries and compositions.
In some embodiments, a delivery vector of the disclosure comprises a composition of the disclosure, such as nucleic acid vector comprising a CBh promoter operably linked to a sequence encoding a GPCR, such as an mGluR. In some embodiments of the delivery vectors of the disclosure, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated vector (AAV). In some embodiments, the AAV is a recombinant AAV (rAAV). In some embodiments, the rAAV comprises a sequence isolated or derived from an AAV of a first serotype and a sequence isolated or derived from an AAV of a second serotype. In some embodiments, the rAAV comprises a capsid sequence isolated or derived from an AAV of a first serotype and a capsid insert sequence isolated or derived from an AAV of a second serotype. In some embodiments, the heterologous capsid insert sequence is neither isolated nor derived from an AAV of any known serotype. In some embodiments, the heterologous capsid insert sequence comprises a random sequence.
Exemplary AAV serotypes include, but are not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and any combination thereof. In some embodiments, an AAV vector of the disclosure comprises a sequence isolated or derived from one or more of AAV2, AAV4, AAV5 and AAV8. In some embodiments, an AAV vector of the disclosure comprises a wild type sequence from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9. In some embodiments, an AAV vector of the disclosure comprises a capsid sequence isolated or derived from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9. In some embodiments, an AAV vector of the disclosure comprises a capsid sequence isolated or derived from AAV2 and AAV4. In some embodiments, an AAV vector of the disclosure comprises a capsid sequence isolated or derived from AAV2 and AAV5. In some embodiments, an AAV vector of the disclosure comprises a capsid sequence isolated or derived from AAV2 and AAV8. In some embodiments, an AAV vector of the disclosure comprises a recombinant or chimeric capsid sequence comprising two or more sequences isolated or derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9.
In certain specific embodiments of the present disclosure, modified adeno-associated vectors (AAV) are used as described in WO2018/022905 and/or WO2021243085A2, the contents of which are incorporated herein by reference in their entireties.
In certain specific embodiments of the present disclosure, modified adeno-associated vectors (AAV) are used as described in any of WO 2012/145601, WO 2018/022905, WO 2019/006182 and/or WO2021243085A2, the contents of which are incorporated herein by reference in their entireties. As certain non-limiting examples, in some cases, a modified AAV comprises a variant AAV capsid protein comprising an insertion of a peptide in the GH loop of the capsid protein, e.g., where the insertion site is within amino acids 570-611 (e.g., between amino acids 587 and 588) of an AAV2 capsid protein, or a corresponding site in another AAV serotype. In some cases, the peptide inserted into the GH loop of the capsid protein comprises the amino acid sequence LGETTRP (SEQ ID NO: 50). As another example, in some cases, a peptide inserted into the GH loop of an AAV capsid protein comprises an amino acid sequence selected from the group consisting of LATTSQNKPA (SEQ ID NO: 51), LAVDGAQRSA (SEQ ID NO: 52), LAKSDQSKPA (SEQ ID NO: 53) and LAANQPSKPA (SEQ ID NO: 54) as described in WO2018/022905.
As another example, in some cases, a peptide inserted into the GH loop of an AAV capsid protein comprises an amino acid sequence selected from the group consisting of LAHQDTTKNS (SEQ ID NO: 55), LAHQDSTKNA (SEQ ID NO: 56), LAHQDATKNA (SEQ ID NO: 57), LALSEATRPA (SEQ ID NO: 58), LAKDETKNSA (SEQ ID NO: 59), LQRGNRQTTTADVNTQ (SEQ ID NO: 60), LQRGNRQATTEDVNTQ (SEQ ID NO: 61), SRTNTPSGTTTQPTLQFSQ (SEQ ID NO: 62) and SKTDTPSGTTTQSRLQFSQ (SEQ ID NO: 63), as described in WO2021243085A2.
In some embodiments, delivery vectors, including AAV vectors, target a retinal cell type. In some embodiments, delivery vectors, including AAV vectors, have a tropism for a retinal cell type. In some embodiments, the retinal cell type is a neuron. In some embodiments, the retinal cell type is a retinal ganglion cell. In some embodiments, the retinal cell type is a horizontal cell. In some embodiments, the retinal cell type is an amacrine cell. In some embodiments, the retinal cell type is a bipolar cell. In some embodiments, the retinal cell type is a photoreceptor cell. In some embodiments, the retinal cell type is not a photoreceptor. Photoreceptor cells include rod cells and cone cells.
In some embodiments, the term “targeting” is meant to describe a specific and/or selective binding to the retinal cell type resulting in higher expression of the composition of the disclosure in that retinal cell type than in any other retinal cell type or non-retinal cell type.
In some embodiments, the cell is a retinal neuron or a progenitor cell thereof. In some embodiments, the progenitor cell is a neural fold cell, an early retinal progenitor cell (RPC), a late RPC, an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), or a retinal pigmented epithelial (RPE) cell. In some embodiments, ESCs of the disclosure are neither isolated nor derived from a human embryo or human tissue.
In some embodiments, a composition of the disclosure may be delivered to a differentiated cell and/or a progenitor cell capable of becoming the differentiated cell type.
Therapeutic Indications
Compositions, vectors, cells and pharmaceutical compositions of the disclosure may be administered as a monotherapy. Alternatively, compositions, vectors, cells and pharmaceutical compositions of the disclosure may be administered as combination therapies.
Compositions, vectors, cells and pharmaceutical compositions of the disclosure may be used for the manufacture of a medicament to treat or may be used in a method for the treatment of a disease or disorder. In some embodiments, the disease or disorder is an ocular disease or disorder. In some embodiments, the disease or disorder is a retinal disease or disorder.
Retinal diseases or disorders may be congenital, degenerative or traumatic. Compositions, vectors, cells and pharmaceutical compositions of the disclosure may be used to restore cellular function or activity to any retinal neuron of an intact or diseased retina. In some embodiments, compositions, vectors, cells and pharmaceutical compositions of the disclosure may be used to restore vision to a subject by inducing a new function or activity to any retinal neuron of an intact or diseased retina to compensate for a missing or lost function or activity in any retinal neuron.
In some embodiments, methods are provided for the enhancement and/or restoration of vision in a subject comprising administering a vector of the present disclosure to a subject in need thereof in order to drive the expression of a fusion polypeptide comprising an affinity tag (e.g., a SNAP tag sequence) and a GPCR (e.g., mGluR2) in the retinal cells of the subject. In related embodiments, in addition to the administration of a vector of the disclosure, a photoswitch conjugate is also administered to the subject before, concurrent with or after administration of the vector. Exemplary photoswitch conjugates for use in such methods can be found described, for example, in WO2019/060785 and WO2021/243086, the contents of which are incorporated herein by reference in their entireties. For example, in some specific embodiments, a vector administered to a subject comprises a CBh promoter operably linked to a SNAP-mGluR2 fusion polypeptide as described herein. In addition, a photoswitch conjugate administered to the subject is a BGAG conjugate. In more particular embodiments, the BGAG conjugate comprises benzylguanine, azobenzene and at least one glutamate ligand. In even more particular embodiments, the BGAG conjugate is a branched BGAG molecule, such as 4X-BGAG (as described in WO2021/243086) or 9X-BGAG (as described in U.S. Provisional Application No. 63/283,022, the content of which is incorporated herein by reference in its entirety).
Every document cited herein, including any cross-referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.
While particular embodiments of the disclosure have been illustrated and described, various other changes and modifications can be made without departing from the spirit and scope of the disclosure. The scope of the appended claims includes all such changes and modifications that are within the scope of this disclosure.
Cynomolgus macaques and African Green monkeys between 3-10 years of age were used. Bilateral intravitreal injections were performed using a 30 g needle to deliver 5.0E+11 viral genomes per eye in a 50 μL volume.
Onset and progression of GFP expression is monitored weekly by confocal scanning laser ophthalmoscopy (cSLO) imaging using the autofluorescence function of the Heidelberg Spectralis HRA/OCT system.
Six to eight weeks after intravitreal injection the primates were euthanized and both eyes (whole globes) were carefully harvested. After enucleation, excess orbital tissue was carefully trimmed and removed. A small (5 mm) slit was made ˜2 mm from the limbus and the whole eye was placed in a vial containing 4% paraformaldehyde (PFA) and incubated at 4° C. overnight. After overnight fixation, the PFA was decanted and replaced with phosphate buffered saline (PBS). The whole eye was dissected to remove the anterior structures (cornea, lens, and ciliary body) and then 4 cuts were made to the posterior eye to enable the tissue to lie nearly flat. A fluorescent dissection microscope was used to visualize GFP expression in the entire retina, by direct fluorescence upon filtered UV excitation. The retinal tissue was then dissected into central and peripheral pieces, separated from the underlying tissues, additionally rinsed in PBS, embedded in agarose, sectioned, mounted on microscope slides, and examined by laser-scanning confocal microscopy. After sectioning 4′,6-diamidino-2-phenylindole (DAPI) was used to label cell nuclei. GFP expression is detected by direct fluorescence. Images are acquired at different magnifications to evaluate transduction in the different cell layers.
For the construct AAV-Var17-CBh-ChrimsonR-GFP, containing a CBh promoter of SEQ ID NO: 1, the following results were obtained.
A. Vectors:
The following vectors were used in rd1 mice and non-human primates:
B. Expression Analyses in NHP Retinas:
Cynomolgus macaques and African Green monkeys between 3-10 years of age were used. Bilateral intravitreal injections of the vectors described above were performed using a 30 g needle to deliver 3.0E+11 to 5.0E+11 viral genomes per eye in a 50 μL volume. Onset and progression of GFP expression was monitored weekly by confocal scanning laser ophthalmoscopy (cSLO) imaging using the autofluorescence function of the Heidelberg Spectralis HRA/OCT system.
Six to eight weeks after intravitreal injection the primates were euthanized and both eyes (whole globes) were carefully harvested. After enucleation, excess orbital tissue was carefully trimmed and removed. A small (5 mm) slit was made ˜2 mm from the limbus and the whole eye was placed in a vial containing 4% paraformaldehyde (PFA) and incubated at 4° C. overnight. After overnight fixation, the PFA was decanted and replaced with phosphate buffered saline (PBS). The whole eye was dissected to remove the anterior structures (cornea, lens, and ciliary body) and then 4 cuts were made to the posterior eye to enable the tissue to lie nearly flat.
A fluorescent dissection microscope was used to visualize GFP expression in the entire retina, by direct fluorescence upon filtered UV excitation. The retinal tissue was then dissected into central and peripheral pieces, separated from the underlying tissues, additionally rinsed in PBS, embedded in agarose, sectioned, mounted on microscope slides, and examined by laser-scanning confocal microscopy. After sectioning, 4′,6-diamidino-2-phenylindole (DAPI) was used to label cell nuclei. GFP expression was detected by direct fluorescence. Images were acquired at different magnifications to evaluate transduction in the different cell layers.
SNAP immunostaining was carried out to visualize SNAP-mGluR2 expression on agarose section. Sections were blocked and permeabilized with Triton-X overnight, incubated with the primary anti-SNAP antibody, rinsed in PBS, incubated with an Alexa-488 conjugated secondary antibody, rinsed, then counterstained with DAPI. Sections were mounted on microscope slides and examined by laser-scanning confocal microscopy.
C. Expression Analyses in Rd1 Mouse Retinas:
Five to seven weeks old rd1 mice were used, which represent a mouse model of blindness due to retinal photoreceptor degeneration. Bilateral intravitreal injections were performed using a 30 g needle to deliver 1.5E+10 viral genomes per eye in a 1.5 μL volume. Six to eight weeks after intravitreal injection of the vectors described above the mice were euthanized and both eyes (whole globes) were carefully harvested. After enucleation, excess orbital tissue was carefully trimmed and removed. A small (5 mm) slit was made ˜2 mm from the limbus and the whole eye was placed in a vial containing 4% paraformaldehyde (PFA) and incubated at 4° C. overnight. After overnight fixation, the PFA was decanted and replaced with phosphate buffered saline (PBS).
The whole eye was dissected to remove the anterior structures (cornea, lens, and ciliary body). For some eyes, the retina was gently detached from posterior eyecup and then 4 cuts were made to enable the tissue to lie nearly flat (“flat-mount”). For some eyes, the posterior eyecup was placed in a 30% sucrose solution overnight to cryoprotect the tissue before embedding in Optimal Cutting Temperature (OCT) medium, freezing and cryosectioning (16 μm cryosections).
A fluorescent dissection microscope was used to visualize GFP expression by direct fluorescence upon filtered UV excitation on retinal flat-mount and cryosections, or SNAP immunostaining was done on retinal flat-mount to visualize SNAP-mGluR2 expression. Retinas were blocked and permeabilized with Triton-X overnight, incubated with the primary anti-SNAP antibody, rinsed in PBS, incubated with an Alexa-488 conjugated secondary antibody, rinsed, then counterstained with DAPI. Sections were mounted on microscope slides and examined by laser-scanning confocal microscopy.
D. Discussion
The results of these studies are shown in
When the vector AAV-Var17-CBh-ChrimsonR-GFP was used in non-human primates, the results shown in
When the vector AAV-Var17-Syn1-ChrimsonR-GFP was used in non-human primates, the results of
When the vector AAV-Var17-CBh-SNAP-mGluR2 was used in non-human primates, the results shown in
When the vector AAV-Var17-Syn1-SNAP-mGluR2 was used in non-human primates, the results shown in
When the vector AAV-Var17-CBh-ChrimsonR-GFP was used in rd1 mice, the results shown in
When the vector AAV-Var17-Syn1-ChrimsonR-GFP was used in rd1 mice, the results shown in
When the vector AAV-Var17-CBh-SNAP-mGluR2 was used in rd1 mice, the results shown in
When the vector AAV-Var17-Syn1-SNAP-mGluR2 was used in rd1 mice, the results shown in
Based on the observation that the synapsin promoter drove much higher levels of expression of the SNAP-mGluR2 transgene in rd1 mice than did the CBh promoter, a similar pattern was expected in the more clinically relevant model system of non-human primates. However, this did not hold true. Instead, unexpectedly, in non-human primates, the CBh promoter drove much more robust expression of a SNAP-mGluR2 transgene in the particular cell types of therapeutic interest in both the central and peripheral retina. These findings make the CBh promoter a highly desirable and advantageous regulatory sequence for driving expression of a heterologous sequence encoding a GPCR (e.g., a SNAP-mGluR2 fusion protein) for addressing ocular disorders and developing vision restoration strategies in humans.
This application is a continuation-in-part of International Application No. PCT/US2022/012019, filed Jan. 11, 2022, which claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/136,144, filed Jan. 11, 2021, each of which is incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63136144 | Jan 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2022/012019 | Jan 2021 | US |
| Child | 17811855 | US |
