AQP1 gene therapy to prevent radiation-induced salivary hypofunction

Abstract

Administration of aquaporin-1 (AQP1) complementary deoxyribonucleic acid (cDNA) to salivary glands prior to treatment with ionizing radiation (IR) prevents subsequent IR-induced loss of function. Administration of AQP1 (e.g., human AQP1; hAQP1) prior to IR treatment (e.g., in head and neck cancer patients) can reduce or prevent IR-induced salivary hypofunction, resulting in increased saliva secretion.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 229,279, filed August 4, 2021, which is incorporated by reference. This patent application also claims the benefit of U.S. Provisional Patent Application No. 63 / 297,342, filed January 7, 2022, which is incorporated by reference.

[0002] Incorporation by Reference of Electronically Submitted Documents The computer readable nucleotide / amino acid sequence listing, filed contemporaneously herewith and identified as follows, is hereby incorporated by reference in its entirety: 6,274 byte file entitled "763111.xml", dated August 4, 2022. [Background technology]

[0003] Ionizing radiation (IR) is a common treatment for patients with head and neck cancer, but administration of IR to these patients can damage the salivary glands, causing irreversible damage and negatively impacting the patient's quality of life.

[0004] There is no conventional treatment for this condition. However, it has been found that administering a vector encoding aquaporin-1 (AQP1) after radiation therapy can affect the repair and redesign of salivary glands. Although this process is effective in some respects, it can cause significant oral health problems in addition to causing significant pain and suffering to patients. Thus, there remains a need for improved methods to treat patients undergoing IR therapy, such as head and neck cancer patients, to counteract the adverse effects of IR therapy on the salivary glands. Summary of the Invention

[0005] In accordance with the present invention, it has surprisingly been found that the detrimental effects of IR therapy can be reduced or prevented by administering a vector encoding an AQP1 prior to irradiation. Administration of AQP1 complementary deoxyribonucleic acid (cDNA) to the salivary glands prior to treatment with IR prevents subsequent IR-induced loss of function. Administration of AQP1 (e.g., human AQP1; hAQP1) prior to IR treatment (e.g., in head and neck cancer patients) can reduce or prevent IR-induced salivary hypofunction, resulting in increased saliva secretion.

[0006] Thus, according to an aspect, the present invention provides vectors (e.g., AAV vectors) encoding AQP1 protein and virions (e.g., AAV virions) comprising such vectors for preventing or reducing radiation-induced salivary dysfunction (e.g., hypofunction) in a subject. Also provided is the use of the vectors or virions for preparing a medicament for preventing or reducing radiation-induced salivary dysfunction in a subject. In one aspect, such vectors or virions are useful for protecting a subject from radiation-induced salivary dysfunction.

[0007] According to an aspect, the present invention provides a method of preventing or alleviating radiation-induced salivary dysfunction in a subject. The method comprises: (a) administering to the subject a vector encoding an aquaporin (AQP) protein; and (b) subsequent to (a), exposing the subject to ionizing radiation, thereby preventing or alleviating radiation-induced salivary dysfunction in the subject. In one aspect, salivary gland function may be maintained at an equivalent or at least equivalent level to salivary gland function prior to administration of ionizing radiation.

[0008] The AQP protein can be any suitable AQP protein, including, but not limited to, an AQP1 protein. For example, in one embodiment, the AQP1 protein is or comprises a human AQP1 (hAQP1) protein.

[0009] The vector encoding the AQP (e.g., hAPQP1) may be or comprise any suitable vector, including, but not limited to, a viral vector. For example, in one embodiment, the viral vector is or comprises an adenoviral vector (e.g., a serotype 2 or serotype 5 adenoviral vector). In another embodiment, the viral vector is or comprises an adeno-associated viral (AAV) vector (e.g., AAV2, AAV5, AAV6, AAV44.9, or BAAV).

[0010] A viral vector (e.g., an AAV vector) may be administered to a subject as a vector or as a virion comprising the vector (e.g., an AAV vector). In one embodiment, the virion is or comprises an AAV virion. The vector or virion may be administered to a subject at any suitable location and by any suitable route of administration. In an embodiment, the vector or virion is administered to the salivary gland of a subject. [Brief description of the drawings]

[0011] [Figure 1] Figure 1 is a graph showing saliva flow rate in mice treated with AQP1. Saliva flow rate is presented in microliters / gm body weight. [Diagram 2] 2 is a graph showing saliva flow rate in mice treated with AQP1. Saliva flow rate is presented in microliters / gm body weight. [Diagram 3] 3 is a graph showing saliva flow rate in mice treated with AQP1. Saliva flow rate is presented in microliters / gm body weight. [Figure 4]Figure 4 shows a heat map of salivary gland cells analyzed by single-cell RNAseq. UMAPs were generated using single-cell RNAseq data from either non-IR mice, GFP-treated IR mice, or mice treated with AQP1 before (AQP1 B) or after (AQP1A), and 16 distinct cell clusters were identified (y-axis). Comparison of cell distribution between the different clusters in each of the four conditions was used to generate heat maps. [Figure 5A-5D] Figures 5A-D are images obtained from histological examination of mouse submandibular glands. Images were taken from whole slides scanned over approximately the same area of ​​each gland near the hilus. The dashed boxes in the 5× sectioned insets are to highlight radiation-induced changes, features including fibrosis, atrophy, and inflammation. Figure 5A corresponds to AAV-GFP before irradiation. Figure 5B corresponds to AAV-AQP1 before irradiation. Figure 5C corresponds to AAV-GFP after irradiation. Figure 5D corresponds to AAV-AQP1 after irradiation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention provides a method comprising pre-treating a subject (e.g., a human patient) undergoing IR treatment affecting a salivary gland (e.g., parotid, submandibular, or sublingual gland) with a vector encoding an aquaporin (AQP) protein. For example, the method may be applied to a subject undergoing IR treatment for cancer, such as head and neck cancer. Thus, according to a first aspect, the present invention provides a method for preventing or reducing radiation-induced salivary dysfunction in a subject, comprising: (a) administering to the subject a vector encoding an aquaporin (AQP) protein; and (b) subsequent to (a), irradiating the subject with ionizing radiation, thereby preventing or reducing radiation-induced salivary dysfunction in the subject.

[0013] As used herein, an aquaporin protein, also referred to as an AQP protein, may be or include any protein that exhibits an activity of an exemplary aquaporin protein (e.g., human aquaporin ("hAQP")), such as the ability to form a channel that allows the passage of water. AQP proteins, nucleic acids, and associated vectors are known to those of skill in the art and are described, by way of non-limiting example, in U.S. Patent No. 10,166,299, which is incorporated herein by reference in its entirety.

[0014] An AQP protein in the context of the present invention may have or include a wild-type (wt) AQP sequence (i.e., having the same amino acid sequence as a naturally occurring AQP protein), may be or include any portion of a wt AQP protein, or may be or include a variant of a naturally occurring AQP protein, provided that such portion or variant retains the ability to form a channel that allows the passage of water. Assays for determining the ability of the AQP proteins of the present invention to form a channel that allows the passage of water are known to those of skill in the art (see, e.g., Lui et al., Journal of Biological Chemistry, 281, 15485-15495 (2006), which is incorporated herein by reference in its entirety).

[0015] In one aspect, the protein useful in the method of the present invention is AQP1 protein, which comprises the full amino acid sequence of naturally occurring AQP1 protein.Examples of human AQP1 protein include, but are not limited to, NCBI reference number NP_932766.1 (SEQ ID NO: 1), NCBI reference number NP_001171989.1 (SEQ ID NO: 2), and NCBI reference number NP_001171990.1 (SEQ ID NO: 3), and NP_001171991.1 (SEQ ID NO: 4).Examples of mouse AQP1 protein include, but are not limited to, SEQ ID NO: 5.

[0016] Examples of AQP proteins, nucleic acids, and associated vectors for use in the present invention are described herein and in US Pat. No. 10,166,299, which are incorporated by reference in their entireties.

[0017] In one embodiment, the AQP1 protein comprises a portion of the amino acid sequence of the AQP1 protein, such portion of the AQP1 protein retains the ability to form a channel in a cell membrane that allows water to pass through. There are several isoforms of the AQP1 protein. Thus, in one embodiment, the AQP1 protein is or comprises an isoform of an AQP protein, such isoform retains the ability to form a channel in a cell membrane that allows water to pass through. In one embodiment, the AQP1 protein is or comprises a portion of an isoform or other naturally occurring variant of the AQP1 protein, such portion retains the ability to form a channel in a membrane that allows water to pass through. Methods for generating functional portions and variants, such as conservative variants, of the AQP1 protein are known to those skilled in the art.

[0018] The present invention also encompasses AQP1 protein variants that have been altered by genetic engineering. For such variants, any kind of change in amino acid sequence is acceptable, as long as the variant retains at least one AQP1 protein activity described herein. Examples of such diversity include, but are not limited to, amino acid deletion, amino acid insertion, amino acid substitution, and combinations thereof. For example, it is well understood by those skilled in the art that one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids can often be removed from the amino and / or carboxy terminus of a protein without significantly affecting the activity of the protein. Similarly, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids can often be inserted into a protein without significantly affecting the activity of the protein.

[0019] As mentioned above, the isolated variant protein of the present invention can also contain amino acid substitutions compared to the wild-type AQP1 protein disclosed herein. Any amino acid substitution is acceptable as long as it does not significantly affect the activity of the protein. In this regard, it is understood in the art that amino acids can be classified into groups based on their physical properties. Examples of such groups include, but are not limited to, charged amino acids, uncharged amino acids, polar uncharged amino acids, and hydrophobic amino acids. A preferred variant that includes substitutions is one in which an amino acid is replaced with an amino acid from the same group. Such substitutions are called conservative substitutions.

[0020] Those skilled in the art can determine the desired amino acid substitution (whether conservative or non-conservative) at the time such substitution is desired. For example, amino acid substitutions can be used to identify important residues of the AQP1 protein or to increase or decrease the affinity of the AQP1 protein described herein. Thus, in one aspect, the AQP1 protein variant comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any range of values ​​therein) amino acid substitution (e.g., conservative substitution) to the AQP1 protein described herein (e.g., SEQ ID NOs: 1-5). In one aspect, the AQP1 protein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an AQP1 protein described herein (e.g., SEQ ID NOs: 1-5).

[0021] Although the proteins of the present invention may consist solely of the sequences disclosed herein and the disclosed variants thereof, such proteins may further contain amino acid sequences that do not confer AQP1 activity but have other useful functions. Any useful additional amino acid sequence may be added to the isolated protein sequence, so long as the additional sequence does not undesirably affect the protein's ability to form a channel that allows the passage of water. For example, the isolated proteins of the present invention may contain amino acid sequences that are useful for visualizing or purifying the peptide. Such sequences act as labels (e.g., enzymes) or tags (e.g., antibody binding sites). Examples of such labels and tags include, but are not limited to, β-galactosidase, luciferase, glutathione-s-transferase, thioredoxin, HIS tags, biotin tags, and fluorescent tags. Other useful sequences for labeling or tagging proteins are known to those of skill in the art.

[0022] In addition to the above modifications, the isolated proteins of the present invention may be further modified, so long as such modifications do not significantly affect the ability of the protein to form a channel that allows the passage of water. Such modifications may be made, for example, to increase the stability, solubility, or absorbability of the protein. Examples of such modifications include, but are not limited to, pegylation, glycosylation, phosphorylation, acetylation, myristylation, palmitoylation, amidation, and / or other chemical modifications of the peptide.

[0023] The AQP1 protein may be from any species that expresses a functional AQP1 protein. The AQP1 protein may comprise the sequence or a portion of the AQP1 protein of a human or other mammal. Further examples include, but are not limited to, the AQP1 protein of a mouse, a cat, a dog, a horse, a cow, a sheep, a pig, or other companion animal, other zoo animal, or other livestock animal. In one embodiment, the AQP1 protein comprises the amino acid sequence or a portion of the human AQP1 protein. In another embodiment, the AQP1 protein comprises the amino acid sequence or a portion of the mouse AQP1 protein.

[0024] In one aspect, the AQP1 protein is joined to a fusion segment, and such a protein is called an AQP1 fusion protein. Such a protein includes an AQP1 protein domain (also referred to herein as an AQP1 domain) and a fusion segment. A fusion segment is an amino acid segment of any size that can improve the properties of an AQP1 protein. For example, a fusion segment of the present invention can increase the stability of an AQP1 fusion protein, add flexibility, or enhance or stabilize the multimerization of an AQP1 fusion protein. Examples of fusion segments include, but are not limited to, immunoglobulin fusion segments, albumin fusion segments, and any other fusion segments that extend the biological half-life of a protein, provide flexibility to a protein, and / or enhance or stabilize multimerization. It is within the scope of the present disclosure to use one or more fusion segments. A fusion segment may be joined to the amino terminus and / or carboxyl terminus of an AQP1 protein of the present invention. As used herein, "joining" refers to combining by linkage using genetic engineering techniques. In such embodiments, a nucleic acid molecule encoding the AQP1 protein is physically linked to a nucleic acid molecule encoding the fusion segment such that the two coding sequences are in frame and the transcription products form a contiguous fusion protein. In one embodiment, the AQP1 protein may be joined directly to the fusion segment or the AQP1 protein may be linked to the fusion segment by a linker of one or more amino acids.

[0025] Nucleic acid molecules (polynucleotides) encoding the AQP1 proteins (e.g., AQP1 fusion proteins) described herein are also provided as aspects of the invention. Nucleic acid molecules may comprise DNA, cDNA, and / or RNA, may be single-stranded or double-stranded, and may be natural, synthetic, and / or recombinant.

[0026] Polynucleotides may contain nucleotide analogs or derivatives, such as inosine or phosphorothioate nucleotides. Silent variations in coding sequences result from the degeneracy (i.e., redundancy) of the genetic code, whereby more than one codon can code for the same amino acid residue. Thus, for example, CTT, CTC, CTA, CTG, TTA, or TTG can code for leucine; TCT, TCC, TCA, TCG, AGT, or AGC can code for serine; AAT or AAC can code for asparagine; GAT or GAC can code for aspartic acid; TGT or TGC can code for cysteine; GCT, GCC, GCA, or GCG can code for alanine; CAA or CAG can code for glutamine; TAT or TAC can code for tyrosine; and ATT, ATC, or ATA can code for isoleucine.

[0027] The polynucleotide may be provided as part of a construct that includes the polynucleotide and elements that allow delivery of the polynucleotide to a cell and / or expression of the polynucleotide in a cell. For example, a polynucleotide sequence encoding AQP1 may be operably linked to an expression control sequence. The expression control sequence operably linked to a coding sequence is ligated such that the coding sequence is expressed under conditions compatible with the expression control sequence. Expression control sequences include, but are not limited to, suitable promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of the gene encoding the protein, splicing signals of introns, maintenance of the correct reading frame of the gene to allow proper translation of the mRNA, and stop codons, etc. Suitable promoters include, but are not limited to, SV40 early promoter, RSV promoter, adenovirus major late promoter, human CMV immediate early type I promoter, poxvirus promoter, 30K promoter, I3 promoter, sE / L promoter, 7.5K promoter, 40K promoter, and C1 promoter.

[0028] Polynucleotides encoding AQP1 or fusion proteins can be cloned or amplified by in vitro methods such as polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification system (TAS), self-sustained sequence replication system (3SR), and QOE≦replicase amplification system (QB). For example, polynucleotides encoding zinc finger proteins can be isolated by polymerase chain reaction of cDNA using primers based on the DNA sequence of the molecule. A wide variety of cloning and in vitro amplification methodologies are well known to those of skill in the art.

[0029] Vectors for use in the present invention include plasmids (e.g., DNA plasmids), bacterial vectors, and viral vectors, such as adenovirus vectors, adeno-associated virus (AAV) vectors, poxvirus vectors, retrovirus vectors, herpesvirus vectors, poliovirus vectors, and alphavirus vectors. When the vector is a plasmid (e.g., a DNA plasmid), the plasmid may be complexed with chitosan.

[0030] In one aspect, the vector is or comprises a viral vector, such as an adenoviral vector (e.g., serotype 2 or serotype 5) or an adeno-associated virus (AAV) vector. Such an AAV vector can be selected from an AAV1 vector, an AAV2 vector, an AAV3 vector, an AAV4 vector, an AAV5 vector, an AAV6 vector, an AAV7 vector, an AAV8 vector, an AAV9 vector, an AAV10 vector, an AAV11 vector, an AAV12 vector, an AAV44.9 (described in U.S. Patent Application Publication No. 2018 / 0355376 and the aforementioned U.S. Patent No. 10,166,299, which are incorporated herein in their entireties), an AAAV vector, and a BAAV vector, any such vector encoding the AQP1 protein described herein.

[0031] In one aspect, the AAV vector is or comprises an AAV2 vector, an AAV5 vector, an AAV6 vector, or a BAAV vector, each of which encodes an AQP1 protein as described herein. In one aspect, the AAV vector comprises an AAV ITR and a CMV promoter operably linked to a nucleic acid molecule encoding the AQP1 protein.

[0032] Also provided is a plasmid vector that encodes AQP1 protein.Such a plasmid vector may also include a control region such as AAV ITR, a promoter that is operably linked to the nucleic acid molecule that encodes AQP1 protein, one or more splice sites, a polyadenylation site, and a transcription termination site.Such a plasmid vector also usually includes multiple restriction enzyme sites together with the nucleic acid molecule that encodes drug resistance.

[0033] The present invention also provides AAV virions.As used herein, AAV virions include the AAV vectors encoding the AQP1 protein of the present invention encapsidated in AAV capsids.Examples of AAV capsids include AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAV9 capsid, AAV10 capsid, AAV11 capsid, AAV12 capsid, AAV44.9 capsid, AAAV capsid, BAAV capsid, and capsids from other AAV serotypes known to those skilled in the art.In one aspect, the capsid is a chimeric capsid, i.e., a capsid that comprises VP proteins from more than one serotype. As used herein, the serotype of the AAV virion of the present invention is the serotype conferred by the VP capsid protein. For example, an AAV2 virion is a virion that contains AAV2 VP1, VP2, and VP3 proteins. As long as the virion can efficiently transduce ductal or acinar cells of the salivary gland, any AAV virion can be used to carry out the method of the present invention.

[0034] In one aspect, the AAV virion is selected from an AAV2 virion, an AAV5 virion, an AAV6 virion, and a BAAV virion, and the AAV vector within the virion encodes an AQP1 protein.

[0035] Methods useful for producing the AAV vectors and AAV virions disclosed herein are known to those skilled in the art. Briefly, the AAV vectors of the present invention can be produced using recombinant DNA or RNA techniques to isolate and join together the nucleic acid sequences of interest as described herein, for example, by using techniques known to those skilled in the art such as restriction enzyme digestion, ligation, PCR amplification, etc. The method for producing the AAV virions of the present invention generally includes (a) introducing the AAV vectors of the present invention into a host, (b) introducing a helper vector into a host cell, the helper vector comprising the viral functions missing from the AAV vector, and (c) introducing a helper virus into the host cell. To achieve replication and packaging of the AAV vector into AAV virions, all functions for replication and packaging of the AAV virions must be present. In some instances, at least one of the viral functions encoded by the helper vector can be expressed by the host cell. The introduction of the vector and the helper virus can be performed using standard techniques and can be performed simultaneously or sequentially. The host cells are then cultured to produce AAV virions, which are then purified using standard techniques, such as CsCl gradients. Residual helper virus activity can be inactivated using known methods, such as heat inactivation. Such methods typically yield high titers of highly purified AAV virions ready for use.

[0036] AAV vectors of a certain serotype can be packaged in the capsid of the same serotype.For example, AAV2 vectors can be packaged in AAV2 capsid.In another example, AAV vectors of a certain serotype are packaged in the capsid of different serotypes to change the tropism of the resulting virion.Those skilled in the art can determine the combination of serotype of AAV vector and serotype of AAV capsid.

[0037] Vectors for use in the present invention may contain an expression control sequence operably linked to a coding sequence such that expression of the coding sequence is achieved under conditions compatible with the expression control sequence, including, but not limited to, a suitable promoter, enhancer, transcription terminator, a start codon (i.e., ATG) in front of the gene encoding the protein, splicing signals for introns, maintenance of the correct reading frame of the gene to allow proper translation of mRNA, and stop codons, etc.

[0038] As used herein, the term "enhancer" refers to a DNA sequence that, for example, increases the transcription of a nucleotide sequence to which it is operably linked. Enhancers may be located many kilobases away from the coding region of a nucleotide sequence and can mediate the binding of regulatory factors, patterns of DNA methylation, or changes in DNA structure. A large number of enhancers from a variety of different sources are known in the art and are available as or within cloned polynucleotides (e.g., from depositories such as the ATCC, as well as other commercial or private sources). Many polynucleotides that contain a promoter (such as the commonly used CMV promoter) also contain enhancer sequences. Enhancers can be located upstream, within, or downstream of a coding sequence. For example, a nucleotide sequence encoding a polypeptide can be operably linked to a CMV enhancer / chicken β-actin promoter (also referred to as a "CAG promoter"). Additionally, a vector can contain a nucleic acid sequence encoding a reporter for determining the transfection / transduction efficiency of the vector.

[0039] Compositions are also provided that include vectors (e.g., AAV vectors) encoding AQP proteins. Compositions are also provided that include AAV virions that include AAV vectors encoding AQP1 proteins. Such compositions can include a carrier (e.g., a pharma- ceutical or physiologically acceptable carrier). For example, such compositions can include an aqueous solution, such as a physiologically compatible buffer. Examples of excipients that can be included in the composition include water, saline, Ringer's solution, and other physiologically balanced salt solutions. In some embodiments, excipients are added, for example, to maintain particle stability or prevent aggregation. Examples of such excipients include, but are not limited to, magnesium to maintain particle stability, pluronic acid to reduce adhesion, mannitol to reduce aggregation, and the like, as known to those skilled in the art.

[0040] The composition is conveniently formulated in a form suitable for administration to a subject. Techniques for formulating such compositions are known to those skilled in the art. For example, the vector (e.g., AAV vector) or virion of the present invention can be combined with saline or other pharmaceutically acceptable solution. In some embodiments, excipients are also added. In another embodiment, the composition containing the vector (e.g., AAV vector) or virion can be dried, and saline or other pharmaceutically acceptable solution can be added to the composition before administration.

[0041] Additionally, the vectors or virions, either alone or as part of a pharmaceutical preparation, can be used in the methods described herein.

[0042] The composition (e.g., pharmaceutical composition) may also contain one or more other additional therapeutic agents. Examples of such additional therapeutic agents that may be suitable for use in the composition include gene therapy agents, anti-inflammatory agents, free radical scavengers, radiation protection agents, and agents or drugs that increase saliva production.

[0043] A viral vector (e.g., an AAV vector) may be administered to a subject as a vector or as a virion comprising the vector (e.g., an AAV vector). In one embodiment, the virion is an AAV virion. The vector or virion may be administered to a subject at any suitable location and by any suitable route of administration. In an embodiment, the vector or virion is administered to the salivary gland of a subject.

[0044] As used herein, the ability of a vector or virion to prevent or reduce radiation-induced salivary dysfunction refers to the ability of such vector or virion to completely or partially eliminate radiation-induced salivary dysfunction. For example, with respect to salivary flow rate, the methods of the invention can restore such flow rate to 70%, 80%, 85%, 90%, 95%, or 100% of the value observed in normal individuals (i.e., individuals not administered radiation).

[0045] The present disclosure provides a method comprising administering a vector or virion to a subject, such administration maintains salivary gland function in such subject after radiation administration. As used herein, maintaining salivary gland function in a subject to which a vector or virion has been administered means that the salivary gland function after radiation administration is equivalent (or at least equivalent) to the salivary gland function in the subject before radiation administration. For example, after irradiation of a subject to which a vector or virion has been administered, the salivary gland function of the subject does not deteriorate and is equivalent (or at least equivalent) to the function before radiation administration. In an embodiment of the present invention, the method comprises (a) administering a vector encoding an AQP protein to a subject not treated with ionizing radiation, and (b) following (a), administering ionizing radiation to the subject, thereby preventing or reducing radiation-induced salivary dysfunction in the subject.

[0046] As used herein, "subject" includes humans and other mammals, such as mice, rats, hamsters, cats, dogs, pigs, cows, horses, other companion animals, other zoo animals, laboratory animals (e.g., mice), and farm animals.

[0047] The vector or virion can be administered by a variety of routes. In some embodiments, the vector or virion is administered by aerosol. In some embodiments, the vector or virion is administered to a mucosa. In some embodiments, the vector or virion is administered directly to a tissue or organ. In some embodiments, the vector or virion is administered to a salivary gland (e.g., parotid, submandibular, or sublingual gland).

[0048] The present invention also provides ex vivo methods for preventing or reducing radiation-induced salivary dysfunction. Such methods may involve administering vectors or virions to a cell, tissue, or organ outside the subject's body and then placing the cell, tissue, or organ in the body. Such methods are known to those of skill in the art.

[0049] In an aspect, the invention provides a cell (e.g., a salivary gland cell), tissue, or organ transfected with an AAV vector encoding an AQP1 protein. The cell (e.g., a salivary gland cell), tissue, or organ (e.g., a salivary gland such as a parotid, submandibular, or sublingual gland) may be from a subject who is planning to be irradiated or has been irradiated, or may be an ex vivo cell, tissue, or organ.

[0050] The vector, virion, or composition thereof (e.g., pharmaceutical composition) can be administered alone or in combination with one or more other additional therapeutic agents. Examples of such additional therapeutic agents that may be suitable include gene therapy agents, anti-inflammatory agents, free radical scavengers, radioprotectors, and agents or drugs that increase saliva production.

[0051] The dose of the compositions disclosed herein administered to a subject to be effective (i.e., to prevent or reduce radiation-induced salivary dysfunction) will depend on the condition of the subject, the method of administration, and the judgment of the prescribing physician. An exemplary dose is about 10 virion particles per kilogram of subject. 4 Approximately 10 virions per kilogram 12can range from 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 pieces, 10 10 pieces, 10 11 pieces, 10 12 A preferred dose is about 10 virion particles per kilogram. 6 Approximately 10 virions per kilogram 12 A more preferred dose is about 10 virion particles per kilogram. 8 Approximately 10 virions per kilogram 12 It is a range of pieces.

[0052] An exemplary dose is about 10 virion particles per gram of gland. 4 Approximately 10 virion particles per gram of gland 12 can range from about 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 pieces, 10 10 pieces, 10 11 pieces, 10 12 (or a range thereof).

[0053] In some embodiments, the dose is determined by the amount of fluid required to fill the gland. For vector-cell contact to occur, the gland must be filled with fluid in order to introduce the vector into the cells. For IR patients, the volume ranges from about 500 μL to about 2.5 mL, depending on atrophy and fibrosis (e.g., as follows: 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, 1 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, 2 mL, 2.1 mL, 2.2 mL, 2.3 mL, 2.4 mL, 2.5 mL, or ranges thereof). EXAMPLES

[0054] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

[0055] Example 1 This example demonstrates that administration of an AQP1-encoding vector (AAV vector) before and after IR treatment promotes salivary gland function.

[0056] All mouse experiments were approved by the National Institute of Dental and Craniofacial Research (NIDCR) Animal Care and Use Committee. Eight-week-old female C3H mice (National Cancer Research Center Animal Experiment Facility) were used. Ten days before or two months after irradiation (IR), groups of seven to eight C3H mice were treated with AAV2 vectors encoding either GFP, AQP1, neurturin, or a combination of AAV2-AQP1 + AAV2-neurturin.

[0057] Specifically, the treatment groups were treated with AAV2-GFP (10 10 vp / g) or AAV2-hAQP1(10 10 or 10 7 vp / g) or AAV2NRTN(10 6 , 10 8 , or 10 10 The study included injecting saliva with 100 mg / kg of 1000 mg ...

[0058] Mice were irradiated (6 Gy / day for 5 days) in five fractions using a Therapax DXT300 X-ray irradiator (Pantak). After IR, animals were removed from the fixture and housed (5 per cage) in a climate- and light-controlled environment with free access to food and water. To deliver the viral vector to the submandibular gland, mice were anesthetized intramuscularly with ketamine (60 mg / kg) and xylazine (8 mg / kg), after which the vector was delivered to both submandibular glands by retroductal injection. During cannulation, 0.5 mg / kg atropine was applied intramuscularly to suppress saliva secretion to increase transduction efficiency.

[0059] To collect saliva, mice were anesthetized as described above and then subcutaneously injected with 0.25 mg pilocarpine per kg body weight to stimulate salivation. Whole saliva was collected for 20 min into 1.5 mL pre-weighed Eppendorf tubes using 75 mm hematocrit tubes (Drummond) and immediately frozen. After 10 months, mice were sacrificed in a carbon dioxide chamber and glands were removed for analysis. Saliva was collected from mice before the start of the experiment (baseline / non-IR). The results are presented in Figures 1-3.

[0060] The results showed that compared to AAV2-GFP-treated mice, AAV2-AQP1 treatment was able to prevent the loss of salivary flow when administered before IR or elicit a recovery of salivary flow after IR (p<.01) (see Figures 1-3). In contrast, neurturin was only able to prevent the loss of salivary flow but was unable to elicit a substantial recovery. Furthermore, the combination of the two vectors had no synergistic effect and substantially increased salivary flow beyond the levels achieved by administering the AAV2AQP1 vector alone before or after IR.

[0061] These results support that administration of AAV-AQP1 before or after IR treatment promotes salivary gland function.

[0062] Prior to the present invention, the conventional understanding was that AQP1 gene therapy required stable epithelial cells to form an enhanced pathway for fluid movement by AQP1 expression. In clinical trials of AQP1, patients were required to be at least 2 years post-IR treatment. Since cell turnover and remodeling after IR treatment is significant, it is expected that AQP1 AAV DNA will be lost (i.e., will not persist) from transduced cells after IR treatment and therefore will not be able to form an enhanced pathway for fluid movement. For example, Malik et al., J. Virol., 71(3):1776-1783 (1997)) teach that AAV can only persist in non-dividing cells and is lost over time in dividing populations. Li et al., Int. J. Radiation Oncology Biol. Phys., 62(5):1510-1516 (2005)) teach that the gland undergoes significant remodeling after IR, with subsequent loss of function. Furthermore, Vitolo et al., Oral Diseases, 8:183-191 (2002) teach that AQP1 gene therapy is for gland repair, whereas other approaches are for preventing IR damage to the gland. Vitolo et al. also disclose that the salivary gland is a slowly dividing cell population, and that AAV transduction in the salivary gland can be sustained.

[0063] Thus, prior to the present invention, AQP1 gene therapy was not considered a preventative approach to IR-induced functional decline because AQP1 gene therapy would not persist in an environment of change and remodeling, such as the salivary gland after IR. However, as described herein, the invention disclosed herein is surprising and unexpected, as it promotes salivary gland function by administering AAV-AQP1 before and after IR treatment.

[0064] Example 2 This example characterizes the mechanisms of saliva flow before and after IR.

[0065] Using single-cell RNAseq data from either non-IR mice, GFP-treated IR mice, or mice treated with AQP1 before (AQP1B) or after (AQP1A), UMAPs were generated and 16 distinct cell clusters were identified. Comparison of cell distribution between the different clusters in each of the four conditions was used to generate the heatmaps in Figure 4.

[0066] AQP1B is found in the same clade as non-IR, whereas GFP and AQP1A are in separate clades from each other and from the AQP1B / non-IR clade. This result suggests that cell populations differ between AQP1A and AQP1B. Furthermore, the clade organization of the cell type-based distribution of AQP1B is most similar to non-IR, whereas AQP1A forms a separate clade from this group and the IR-effect GFP group.

[0067] These results confirm that when administered prior to IR, salivary flow is the result of glandular protection, but when administered after IR, restoration of salivary flow is possible in a different cell population and environment.

[0068] Example 3 This example demonstrates that administration of AAV-AQP1 before irradiation results in less pathological changes than administration of AAV-AQP1 after irradiation.

[0069] Histological evaluation of mouse submandibular glands was performed. Images of mice administered AAV-GFP before irradiation (Figure 5A), mice administered AAV-AQP1 before irradiation (Figure 5B), mice administered AAV-GFP after irradiation (Figure 5C), and mice administered AAV-AQP1 after irradiation (Figure 5D) were taken from whole slides scanned over roughly the same area of ​​each gland near the hilus. Overall morphological changes in the glands were evaluated by H&E staining.

[0070] Sections were assessed for atrophy, fibrosis, and immune infiltration using a scoring scale ranging from 0 to 3. Overall, all showed increased atrophy and fibrosis. There was also increased inflammation with intraglandular germinal center formation and the presence of reactive lymph nodes within the glandular capsule. There was also an increased number of multinucleated acinar cells and some areas of ductal hyperplasia.

[0071] However, the mean score of the post-treatment group was 2.2 ± 0.75 points, whereas that of the pre-treatment group was 1.5 ± 0.54 points (p > 0.05). Thus, the post-IR treatment group tended to show more pronounced pathological changes compared with the pre-IR treatment group (Figure 5A-D).

[0072] These results confirm that administration of an AQP1-encoding vector before irradiation mitigates the deleterious effects observed when an AQPI-encoding vector is administered after irradiation.

[0073] All references cited in this specification, including publications, patent applications, and patents, are hereby incorporated by reference as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0074] Use of the terms "a" and "an" and "the" and "at least one" and similar referents in connection with the description of the present invention (particularly in connection with the claims that follow) should be construed to cover both the singular and the plural, unless otherwise specified herein or clearly contradicted by context. Use of the term "at least one" following a list of one or more items (e.g., "at least one of A and B") should be construed to mean one item (A or B) selected from the listed items or any combination of two or more of the listed items (A and B), unless otherwise specified herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including but not limited to"), unless otherwise specified herein or clearly contradicted by context. The recitation of ranges of values ​​herein is intended to serve merely as a shorthand for referring individually to each separate value within the range, and each separate value is incorporated into the specification as if it were individually recited herein, unless otherwise specified herein. All methods described herein can be performed in any suitable order, unless otherwise specified herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to further elucidate the invention, and does not pose a limitation on the scope of the invention, unless otherwise claimed. No language in the specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0075] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of the preferred embodiments may become apparent to those of skill in the art upon reading the foregoing description. The inventors expect that such variations will be utilized by those of skill in the art, and the inventors intend that the invention be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context.

[0076] Biological sequences The following sequences are referenced herein: SEQ ID NO:1: 1 masefkkklf wravvaefla ttlfvfisig salgfkypvg nnqtavqdnv kvslafglsi 61 atlaqsvghi sgahlnpavt lglllscqis ifralmyiia qcvgaivata ilsgitsslt 121 gnslgrndla dgvnsgqglg ieiigtlqlv lcvlattdrr rrdlggsapl aiglsvalgh 181 llaidytgcg inparsfgsa vithnfsnhw ifwvgpfigg alavliydfi laprssdltd 241 rvkvwtsgqv eeydldaddi nsrvemkpk SEQ ID NO:2: 1 mpgarplplv lvpqntlawm qldakapahp rplqllgrvg pgsrqladgv nsgqglgiei 61 igtlqlvlcv lattdrrrrd lggsaplaig lsvalghlla idytgcginp arsfgsavit 121 hnfsnhwifw vgpfiggala vliydfilap rssdltdrvk vwtsgqveey dldaddinsr 181 vemkpk SEQ ID NO:3: 1 mfwtfgyeav spagpshlfa sllllgvllti tfmpgarplp lvlvpqntla wmqldakapa 61 hprplqllgr vgpgsrqlad gvnsgqglgi eiigtlqlvl cvlattdrrr rdlggsapla 121 iglsvalghl laidytgcgi nparsfgsav ithnfsnhwi fwvgpfigga lavliydfil 181 aprssdltdr vkvwtsgqve eydldaddin srvemkpk SEQ ID NO:4: 1 mqsgmgwnvl dfwladgvns gqglgieiig tlqlvlcvla ttdrrrrdlg gsaplaigls 61 valghllaid ytgcginpar sfgsavithn fsnhwifwvg pfiggalavl iydfilaprs 121 sdltdrvkvw tsgqveeydl daddinsrve mkpk SEQ ID NO:5 1 maseikkklf wravvaefla mtlfvfisig salgfnyple rnqtlvqdnv kvslafglsi 61 atlaqsvghi sgahlnpavt lglllscqis ilravmyiia qcvgaivata ilsgitsslv 121 dnslgrndla hgvnsgqglg ieiigtlqlv lcvlattdrr rrdlggsapl aiglsvalgh 181 llaidytgcs inparsfgsa vltrnfsnhw ifwvgpfigg alavliydfi laprssdftd 241 rmkvwtsgqv eeydldaddi nsrvemkpk

Claims

1. 1. An agent for preventing or alleviating radiation-induced salivary dysfunction in a subject, comprising: an adeno-associated virus (AAV) vector encoding an aquaporin (AQP) protein; The agent is administered to the subject prior to the administration of ionizing radiation.

2. The method of claim 1, wherein the AAV vector is administered to the salivary glands of the subject.

3. The agent according to claim 1 , wherein the AQP protein comprises an AQP1 protein.

4. The agent according to claim 3 , wherein the AQP1 protein comprises a human AQP1 protein.

5. The agent according to any one of claims 1 to 4, wherein the AAV vector is an AAV2, AAV5, AAV6, AAV44.9, or BAAV vector.

6. The agent according to any one of claims 1 to 4, wherein the AAV vector is administered as a virion containing the AAV vector.

7. The method of claim 6, wherein the virion is an AAV2 virion, an AAV5 virion, an AAV6 virion, or a BAAV virion.

8. The agent according to any one of claims 1 to 4, wherein the function of the salivary glands is maintained at a level equivalent to or at least equivalent to the function of the salivary glands before the administration of the ionizing radiation.

9. The agent according to any one of claims 1 to 4, wherein the subject is a human patient.

10. The method of claim 9 , wherein the human patient has head and neck cancer.

11. Use of an AAV vector encoding an aquaporin (AQP) protein in the manufacture of an agent for preventing or alleviating radiation-induced salivary dysfunction in a subject, wherein the agent is administered to the subject prior to administration of ionizing radiation.

12. The use described in claim 11, wherein the AAV vector is administered to the salivary glands of the subject.

13. The use described in claim 11, wherein the AQP protein includes an AQP1 protein.

14. The use described in claim 13, wherein the AQP1 protein comprises human AQP1 protein.

15. The use described in any one of claims 11 to 14, wherein the AAV vector is an AAV2, AAV5, AAV6, AAV44.9, or BAAV vector.

16. The use of any one of claims 11 to 14, wherein the AAV vector is administered as a virion containing the AAV vector.

17. The use of claim 16, wherein the virion is an AAV2 virion, an AAV5 virion, an AAV6 virion, or a BAAV virion.

18. The use of any one of claims 11 to 14, wherein the function of the salivary glands is maintained at a level equivalent to or at least equivalent to the function of the salivary glands before administration of the ionizing radiation.

19. The use described in any one of claims 11 to 14, wherein the subject is a human patient.

20. The use of claim 19, wherein the human patient has head and neck cancer.

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