CN117940147A - AQP1 gene therapy for preventing radiation-induced hyposalivation - Google Patents
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Abstract
The administration of aquaporin-1 (AQP 1) complementary deoxyribonucleic acid (cDNA) to salivary glands prior to treatment of the salivary glands with Ionizing Radiation (IR) prevents subsequent IR-induced loss of function. Administration of AQP1 (e.g., human AQP1; hAQP 1) prior to IR treatment (e.g., in a patient with head and neck cancer) can reduce or prevent IR-induced hyposalivation, resulting in increased salivary output.
Description
Cross Reference to Related Applications
This patent application claims the benefit of U.S. provisional patent application No.63/229,279, filed 8/4 of 2021, which is incorporated herein by reference. The present patent application also claims the benefit of U.S. provisional patent application No.63/297,342, filed on 7/1/2022, which is incorporated herein by reference.
Electronically filed material is incorporated herein by reference
A list of computer-readable nucleotide/amino acid sequences filed concurrently with the present application is incorporated herein by reference and identified as follows: 2022, 8, 4, a 6,274 byte file named "763111. Xml".
Background
A common treatment for patients with head and neck cancer involves Ionizing Radiation (IR). However, administration of IR to these patients can damage the salivary glands, which suffer irreversible damage, negatively affecting the quality of life of the patient.
There is no conventional therapy for this condition. However, it has been found that administration of a carrier encoding aquaporin-1 (AQP 1) after radiation therapy can affect salivary gland repair and remodeling. While effective in some aspects, this procedure can create considerable pain and distress to the patient, as well as significant oral health problems. Thus, there remains a need for an improved method of treating patients undergoing IR treatment, such as head and neck cancer patients, to resist the negative effects of IR treatment on salivary glands.
Disclosure of Invention
In accordance with the present invention, it has surprisingly been found that by applying a vector encoding AQPI prior to irradiation, the deleterious effects of IR treatment can be reduced or prevented. The administration of AQP1 complementary deoxyribonucleic acid (cDNA) to salivary glands prior to treatment with IR prevents subsequent IR-induced loss of function. Administration of AQP1 (e.g., human AQP1; hAQP 1) prior to IR treatment (e.g., in a patient with head and neck cancer) can reduce or prevent IR-induced hyposalivation, resulting in increased salivary output.
Thus, according to one aspect, the present invention provides a vector (e.g., an AAV vector) encoding an AQP1 protein for preventing or reducing radiation-induced saliva dysfunction (e.g., hypofunction) in a subject, and virosomes (e.g., AAV virosomes) comprising such a vector. Also provided is the use of a vector or virosome in the manufacture of a medicament for preventing or alleviating radiation-induced saliva dysfunction in a subject. In one aspect, such vectors or virions can be used to protect a subject from radiation-induced saliva dysfunction.
According to one aspect, the present invention provides a method of preventing or alleviating radiation-induced saliva dysfunction in a subject. The method comprises (a) administering to the subject a carrier encoding Aquaporin (AQP), and (b) administering ionizing radiation to the subject after (a), thereby preventing or alleviating radiation-induced saliva dysfunction in the subject. In one aspect, salivary gland function may be maintained at a level equal to or at least equal to salivary gland function prior to administration of the ionizing radiation.
The AQP protein may be any suitable AQP protein including, but not limited to, AQP1 protein. For example, in one aspect, the AQP1 protein is a human AQP1 (hAQP 1) protein or comprises a human AQP1 (hAQP) protein.
The vector encoding AQP (e.g., hAPQP a) may be or include any suitable vector, including but not limited to viral vectors. For example, in one aspect, the viral vector is or includes an adenovirus vector (e.g., a serotype 2 or serotype 5 adenovirus vector). In another aspect, the viral vector is or includes an adeno-associated virus (AAV) vector (e.g., AAV2, AAV5, AAV6, AAV44.9, or BAAV).
Viral vectors (e.g., AAV vectors) may be administered to a subject as vectors or as virions including vectors (e.g., AAV vectors). In one aspect, the virosome is or includes an AAV virosome. The vector or virosome may be administered to the subject at any suitable location and by any suitable route of administration. In one aspect, the vector or virosome is administered to a salivary gland of a subject.
Drawings
FIG. 1 is a graph showing saliva flow in AQP1 treated mice. Saliva flow is expressed in microliters per gram of body weight.
Fig. 2 is a graph showing saliva flow in AQP1 treated mice. Saliva flow is expressed in microliters per gram of body weight.
FIG. 3 is a graph showing saliva flow in AQP1 treated mice, expressed in microliters per gram of body weight.
FIG. 4 is a heat map of salivary gland cells analyzed by single cell RNAseq. Data from IR mice that were not IR, GFP-treated or single cell RNAseq before AQP1 treatment (AQP 1B) or after AQP1 treatment (AQP 1A) were used to generate UMAP and identify 16 different cell clusters (y-axis). Cell distributions between different clusters under each of the 4 conditions were compared for generating a heat map.
Fig. 5A to 5D are histological images from the mouse submandibular gland. Images were taken from the entire scan slide of the same region of each gland, typically near the gate. The dashed box on 5X defines the inset to emphasize features including radiation induced changes, fibrosis, atrophy and inflammation. FIG. 5A corresponds to AAV-GFP prior to irradiation. FIG. 5B corresponds to AAV-AQP1 prior to irradiation. FIG. 5C corresponds to AAV-GFP after irradiation. FIG. 5D corresponds to AAV-AQP1 after irradiation.
Detailed Description
The present invention provides methods that involve pre-treating a subject (e.g., a human patient) undergoing IR treatment with a carrier encoding Aquaporin (AQP) to affect salivary glands (e.g., parotid, submandibular, or sublingual glands). For example, the method may be applied to a subject undergoing IR treatment for cancer (e.g., head and neck cancer). Thus, according to a first aspect, the present invention provides a method of preventing or alleviating radiation-induced saliva dysfunction in a subject, comprising: (a) Administering to the subject a carrier encoding Aquaporin (AQP), and (b) administering ionizing radiation to the subject after (a), thereby preventing or reducing radiation-induced saliva dysfunction in the subject.
Aquaporins, also referred to herein as AQP proteins, may be any protein that exhibits the activity of an exemplary aquaporin (e.g., human aquaporin ("hAQP")) or include any protein that exhibits the activity of an exemplary aquaporin, thereby forming a channel that allows water to pass through. AQP proteins, nucleic acids, and related vectors are known to those of skill in the art and are described by way of non-limiting example in U.S. patent 10,166,299, which is incorporated herein by reference in its entirety.
In the context of the present invention, an AQP protein may have or comprise a wild-type (wt) AQP sequence (i.e., it has the same amino acid sequence as the native AQP protein), may be any portion of a wild-type AQP protein or comprise any portion of a wild-type AQP protein, or may be a variant of a native AQP protein or comprise a variant of a native AQP protein, provided that such portion or variant retains the ability to form a channel that allows water to pass through. Assays to determine the ability of the AQP proteins of the present invention to form channels that allow water to pass through are known to those of skill in the art (see, e.g., lui et al, journal of Biological Chemistry,281,15485-15495 (2006), the entire contents of which are incorporated herein by reference).
In one aspect, the protein useful in the methods of the invention is an AQP1 protein comprising the entire amino acid sequence of a naturally occurring AQP1 protein. Examples of human AQP1 proteins include, but are not limited to, NCBI reference NP-932766.1 (SEQ ID NO: 1), NCBI reference NP-001171989.1 (SEQ ID NO: 2), and NCBI reference NP-001171990.1 (SEQ ID NO: 3) and NP-001171991.1 (SEQ ID NO: 4). Examples of murine AQP1 proteins include, but are not limited to, SEQ ID NO 5.
Examples of AQP proteins, nucleic acids and related vectors for use in the present invention are described herein and in U.S. patent 10,166,299, the entire contents of which are incorporated herein by reference.
In one aspect, the AQP1 protein comprises a portion of the amino acid sequence of the AQP1 protein, wherein the portion of the AQP1 protein retains the ability to form a channel in the cell membrane that allows water to pass through. There are several subtypes of AQP1 proteins. Thus, in one aspect, the AQP1 protein is or comprises a subtype of AQP-protein, wherein such subtype retains the ability to form channels that allow water to pass through. In one aspect, the AQP1 protein is or comprises a portion of a subtype of AQP1 protein or other naturally occurring variant, wherein the portion retains the ability to form a channel in the membrane that allows water to pass through. Methods for producing functional portions and variants (e.g., conservative variants) of the AQP1 protein are known to those of skill in the art.
The invention also includes AQP1 protein variants altered by genetic manipulation. For such variants, any type of alteration in the amino acid sequence is permissible, provided that the variant retains at least one of the AQP1 protein activities described herein. Examples of such variants include, but are not limited to, amino acid deletions, amino acid insertions, amino acid substitutions, and combinations thereof. For example, it is well known to those skilled in the art that one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids can generally 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 may be inserted in a protein generally without significantly affecting the activity of the protein.
As noted above, the individual variant proteins of the present invention may also contain amino acid substitutions as compared to the wild-type AQP1 proteins disclosed herein. Any amino acid substitution is permissible as long as the activity of the protein is not significantly affected. In this regard, it is understood in the art that amino acids may be categorized 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. Preferred variants containing substitutions are variants in which the amino acids are substituted with amino acids from the same group. Such substitutions are referred to as conservative substitutions.
The desired amino acid substitutions (whether conservative or non-conservative) may be determined by one of ordinary skill in the art when such substitutions are desired. For example, amino acid substitutions can be used to identify important residues of the AQP1 proteins, or to increase or decrease the affinity of the AQP1 proteins described herein. Thus, in one aspect, a variant of an AQP1 protein 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 thereof) amino acid substitution (e.g., conservative substitution relative to an AQP1 protein described herein (e.g., SEQ ID NOs: 1-5). In one aspect, the AQP1 protein comprises an amino acid sequence having 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% identity to an AQP1 protein described herein (e.g., SEQ ID NOS: 1-5).
While the proteins of the present invention may consist entirely of the sequences disclosed herein and variants thereof disclosed, such proteins may additionally 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 individual protein sequences as long as the additional sequence does not have an undesirable effect on the ability of the protein to form a channel that allows water to pass through. For example, the individual proteins of the invention may contain amino acid sequences for visualizing or purifying the peptide. Such sequences are used as labels (e.g., enzymes) or tags (e.g., antibody binding sites). Examples of such labels and tags include, but are not limited to, beta-galactosidase, luciferase, glutathione-s-transferase, thioredoxin, HIS tag, biotin tag, and fluorescent tag. Other useful sequences for labeling and tagging proteins are known to those skilled in the art.
In addition to the modifications described above, the individual proteins of the invention may be further modified, provided that such modifications do not significantly affect the ability of the protein to form channels that allow water to pass through. For example, such modifications may be made to increase the stability, solubility or absorbability of the protein. Examples of such modifications include, but are not limited to, PEGylation, glycosylation, phosphorylation, acetylation, myristoylation, palmitoylation, amidation, and/or other chemical modifications of the peptide.
The AQP1 protein may be derived from any species that expresses a functional AQP1 protein. The AQP1 protein may comprise the sequence of a human or other mammalian AQP1 protein or a portion of the sequence thereof. Other examples include, but are not limited to, murine, feline, canine, equine, bovine, ovine, porcine, or other companion animals, other zoo animals, or other livestock AQP1 proteins. In one aspect, the AQP1 protein comprises the amino acid sequence of a human AQP1 protein or a portion of the sequence thereof. In another aspect, the AQP1 protein comprises the amino acid sequence of a murine AQP1 protein or a portion of the sequence thereof.
In one aspect, the AQP1 protein is linked to a fusion fragment; this protein is known as the AQP1 fusion protein. Such proteins include AQP1 protein domains (also referred to herein as AQP1 domains) and fusion fragments. The fusion fragment is an amino acid fragment of any size that enhances the properties of the AQP1 protein. For example, the fusion fragments of the invention may increase stability, increase flexibility, or enhance or stabilize multimerization of the AQP1 fusion protein. Examples of fusion fragments include, but are not limited to, immunoglobulin fusion fragments, albumin fusion fragments, and any other fusion fragments that increase the biological half-life of a protein, provide flexibility to a protein, and/or achieve or stabilize multimerization. The use of one or more fusion fragments is within the scope of the invention. The fusion fragment may be linked to the amino-and/or carboxy-terminus of the AQP1 proteins of the present invention. As used herein, "ligating" refers to combining by ligation using genetic engineering techniques. In this aspect, the nucleic acid molecule encoding the AQP1 protein is physically linked to the nucleic acid molecule encoding the fusion fragment such that the two coding sequences are in frame and the transcription products form a contiguous fusion protein. In one aspect, the AQP1 protein can be directly linked to the fusion fragment, or the AQP1 protein can be linked to the fusion fragment through a linker of one or more amino acids.
Nucleic acid molecules (polynucleotides) encoding the AQP1 proteins described herein (e.g., AQP1 fusion proteins) are also provided as one aspect of the invention. The nucleic acid molecule may comprise DNA, cDNA and/or RNA, may be single-stranded or double-stranded, and may be naturally occurring, synthetic and/or recombinant.
Polynucleotides may include nucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides, etc.). Silent mutations in the coding sequence result from the degeneracy (i.e., redundancy) of the genetic code, whereby more than one codon can encode the same amino acid residue. Thus, for example, leucine may be encoded by CTT, CTC, CTA, CTG, TTA or TTG; serine can be encoded by TCT, TCC, TCA, TCG, AGT or AGC; asparagine may be encoded by AAT or AAC; aspartic acid may be encoded by GAT or GAC; cysteine may be encoded by TGT or TGC; alanine may be encoded by GCT, GCC, GCA or GCG; glutamine can be encoded by CAA or CAG; tyrosine may be encoded by TAT or TAC; isoleucine may be encoded by ATT, ATC or ATA.
The polynucleotide may be provided as part of a construct comprising the polynucleotide and an element capable of delivering the polynucleotide to and/or expressing the polynucleotide in a cell. For example, the AQP1 encoding polynucleotide sequence may be operably linked to an expression control sequence. The expression control sequences operably linked to the coding sequences are linked such that expression of the coding sequences is effected under conditions compatible with the expression control sequences. Expression control sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, start codons prior to the protein encoding gene (i.e., ATG), splicing signals for introns, maintenance of the correct reading frame of the gene to allow for proper translation of mRNA and stop codons. Suitable promoters include, but are not limited to, the SV40 early promoter, the RSV promoter, the adenovirus major late promoter, the human CMV immediate early I promoter, the poxvirus promoter, the 30K promoter, the I3 promoter, the sE/L promoter, the 7.5K promoter, the 40K promoter, and the C1 promoter.
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 (3 SR) and QAnd (3) a replicase amplification system (QB). For example, polynucleotides encoding zinc finger proteins can be isolated by polymerase chain reaction of cdnas using primers based on molecular DNA sequences. Various cloning and in vitro amplification methods are well known to those skilled in the art.
Vectors useful in the present invention include plasmids (e.g., DNA plasmids), bacterial vectors, and viral vectors (e.g., adenovirus vectors), adeno-associated virus (AAV) vectors, poxvirus vectors, retroviral 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.
In one aspect, the vector is or includes a viral vector, such as an adenovirus vector (e.g., serotype 2 or serotype 5) or an adeno-associated virus (AAV) vector. Such AAV vectors may be selected from AAV1 vectors, AAV2 vectors, AAV3 vectors, AAV4 vectors, AAV5 vectors, AAV6 vectors, AAV7 vectors, AAV8 vectors, AAV9 vectors, AAV10 vectors, AAV11 vectors, AAV12 vectors, and AAV44.9 (as described in U.S. patent application publication No. 2018/0355376), which is incorporated herein in its entirety, and in the aforementioned U.S. patent 10,166,299, AAAV vectors, and BAAV vectors, wherein any of the vectors encodes an AQP1 protein described herein.
In one aspect, the AAV vector is or comprises an AAV2 vector, an AAV5 vector, an AAV6 vector, or BAAV vector, or an AAV2 vector, an AAV5 vector, an AAV6 vector, or BAAV vector, wherein each vector encodes an AQP1 protein described herein. In one aspect, the AAV vector comprises an AAV ITR and a CMV promoter operably linked to a nucleic acid molecule encoding an AQP1 protein.
Plasmid vectors encoding the AQP1 proteins are also provided. Such plasmid vectors may also include a control region (e.g., AAV ITRs), a promoter operably linked to a nucleic acid molecule encoding an AQP1 protein, one or more splice sites, a polyadenylation site, and a transcription termination site. Such plasmid vectors also typically include a number of restriction enzyme sites and nucleic acid molecules encoding drug resistance.
The invention also provides AAV virions. As used herein, AAV virions include AAV vectors encoding the AQP1 proteins of the present invention, which are encapsulated in AAV capsids. Examples of AAV capsids include AAV1 capsids, AAV2 capsids, AAV3 capsids, AAV4 capsids, AAV5 capsids, AAV6 capsids, AAV7 capsids, AAV8 capsids, AAV9 capsids, AAV10 capsids, AAV11 capsids, AAV12 capsids, AAV44.9 capsids, AAAV capsids, BAAV capsids, and capsids from other AAV serotypes known to those of skill in the art. In one aspect, the capsid is a chimeric capsid, i.e., a capsid comprising VP proteins from more than one serotype. As used herein, the serotype of an AAV virion of the invention is the serotype conferred by the VP capsid protein. For example, AAV2 virions are virions that include AAV2 VP1, VP2, and VP3 proteins. Any AAV virion can be used to practice the methods of the invention, provided that the virion is effective to transduce a duct or acinar cell of a salivary gland.
In one aspect, the AAV virion is selected from the group consisting of AAV2 virion, AAV5 virion, AAV6 virion, and BAAV virion, wherein the AAV vector within the virion encodes an AQP1 protein.
Methods for producing the AAV vectors and AAV virions disclosed herein are known to those of skill in the art. Briefly, AAV vectors of the invention can be prepared using recombinant DNA or RNA techniques to isolate nucleic acid sequences of interest and ligate them together as described herein, for example, by using techniques known to those skilled in the art, such as restriction enzyme digestion, ligation, PCR amplification, and the like. Methods of producing AAV virions of the invention generally comprise (a) introducing an AAV vector of the invention into a host, (b) introducing a helper vector into a host cell, wherein the helper vector comprises viral functions deleted from the AAV vector, and (c) introducing a helper virus into the host cell. All functions for AAV virion replication and packaging need to exist to achieve AAV vector replication and packaging into AAV virions. In some cases, at least one viral function encoded by the helper vector may be expressed by the host cell. The introduction of the vector and helper virus may be performed using standard techniques, either 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 may be inactivated using known methods, such as heat inactivation. This approach typically produces highly purified AAV virions at high titers ready for use.
AAV vectors of a particular serotype may be packaged in capsids of the same serotype. For example, an AAV2 vector may be packaged in an AAV2 capsid. In other cases, AAV vectors of a particular serotype are packaged into capsids of different serotypes to alter the tropism of the resulting virions. Combinations of AAV vector serotypes and AAV capsid serotypes can be determined by one of skill in the art.
The vectors used in the present invention may include an expression control sequence operably linked to the coding sequence such that expression of the coding sequence is effected under conditions compatible with the expression control sequence. Expression control sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, initiation codons (i.e., ATGs) prior to the protein encoding gene, splicing signals for introns, maintenance of the correct reading frame of the gene to allow for proper translation of the mRNA, and stop codons.
The term "enhancer" as used herein refers to a DNA sequence that increases transcription of, for example, a nucleotide sequence to which it is operably linked. Enhancers can be located thousands of bases away from the coding region of a nucleotide sequence and can mediate binding of regulatory factors, changes in DNA methylation patterns, or DNA structure. Numerous enhancers from a variety of different sources are well known in the art and are available as or within cloned polynucleotides (e.g., libraries such as ATCC as well as other commercial or personal sources). Many polynucleotides that include a promoter (such as the commonly used CMV promoter) also include enhancer sequences. Enhancers may be located upstream, internal or downstream of the coding sequence. For example, the nucleotide encoding the polypeptide may be operably linked to a CMV enhancer/chicken β -actin promoter (also referred to as a "CAG promoter"). In addition, the vector may include a nucleic acid sequence encoding a reporter gene to identify the transfection/transduction efficiency of the vector.
Also provided are compositions comprising vectors (e.g., AAV vectors) encoding AQP proteins. Also provided are compositions comprising AAV virions comprising an AAV vector encoding an AQP1 protein. Such compositions may include a carrier (e.g., a pharmaceutically or physiologically acceptable carrier). For example, such compositions may include aqueous solutions, such as physiologically compatible buffers. Examples of excipients included in the composition include water, saline, ringer's solution, and other physiologically balanced salt solutions. In some aspects, 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 sticking, mannitol to reduce aggregation, and the like, as known to those skilled in the art.
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 vectors (e.g., AAV vectors) or virions of the invention can be combined with physiological saline or other pharmaceutically acceptable solutions. In some aspects, excipients are also added. In another aspect, the composition comprising the vector (e.g., AAV vector) or virosome is dried and physiological saline or other pharmaceutically acceptable solution may be added to the composition prior to administration.
Furthermore, the vector or virosome may be used in the methods described herein alone or as part of a pharmaceutical formulation.
The composition (e.g., a pharmaceutical composition) may include one or more additional therapeutic agents. Examples of such additional therapeutic agents that may be suitable for use in the composition include gene therapy, anti-inflammatory agents, free radical scavengers, radioprotectors, and agents or drugs that increase saliva production.
Viral vectors (e.g., AAV vectors) may be administered to a subject as vectors or as virions including vectors (e.g., AAV vectors). In one aspect, the virosome is an AAV virosome. The vector or virosome may be administered to the subject at any suitable location and by any suitable route of administration. In one aspect, the vector or virosome is administered to a salivary gland of a subject.
As used herein, the ability of a vector or virion to prevent or reduce radiation-induced saliva dysfunction refers to the ability of such a vector or virion to completely or partially eliminate radiation-induced saliva dysfunction. For example, for saliva flow, the methods of the invention can restore such flow to 70%, 80%, 85%, 90%, 95% or 100% of the value observed in normal individuals (i.e., individuals not administered radiation).
Provided herein is a method comprising administering a vector or virosome to a subject, wherein administration maintains salivary gland function in the subject after irradiation. As used herein, maintaining salivary gland function in a subject to which a vector or virosome has been administered means that the salivary gland function is equivalent (or at least equivalent) to that in the subject prior to administration of radiation after administration of the radiation. For example, after irradiation of a subject to which a vector or virosome has been administered, the subject's salivary gland function is not deteriorated, but is functionally equivalent (or at least equivalent) to that before irradiation. In one aspect of the invention, a method comprises: (a) Applying a vector encoding an AQP protein to a subject not treated with ionizing radiation, and (b) applying ionizing radiation to the subject after (a), thereby preventing or alleviating radiation-induced saliva dysfunction in the subject.
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 livestock.
Vectors or virions can be administered in a variety of ways. In some aspects, the vector or virion is administered by aerosol. In some aspects, the vector or virosome is administered to the mucosa. In some aspects, the vector or virion is administered directly to a tissue or organ. In some aspects, the vector or virosome is administered to a salivary gland (e.g., parotid, submandibular, or sublingual gland).
The invention also provides ex vivo methods of preventing or reducing radiation-induced saliva dysfunction. Such methods may comprise administering the vector or virion to a cell, tissue or organ in vitro of a subject and then placing the cell, tissue or organ in vivo. Such methods are known to those skilled in the art.
In various aspects, the invention provides cells (e.g., salivary gland cells), tissues, or organs transfected with an AAV vector encoding an AQP1 protein. The cell (e.g., salivary gland cell), tissue or organ (e.g., salivary gland, such as parotid gland, submandibular gland, or sublingual gland) may be a subject intended to receive or have received radiation ex vivo, or an ex vivo cell, tissue or organ.
The vector, virosome, or a composition thereof (e.g., a pharmaceutical composition) may be administered alone or in combination with one or more additional therapeutic agents. Examples of suitable such additional therapeutic agents include gene therapy, anti-inflammatory agents, free radical scavengers, radioprotectors, and agents or drugs that increase saliva production.
The dosage of the compositions disclosed herein that will be administered to a subject effective (i.e., preventing or reducing radiation-induced saliva dysfunction) will depend on the condition of the subject, the mode of administration, and the discretion of the prescribing physician. Exemplary dosages may be from about 10 4 virosome particles per kilogram of subject to about 10 12 virosome particles per kilogram of subject (e.g., ,104、105、106、107、108、109、1010、1011、1012 and ranges thereof). A preferred dosage range is from about 10 6 virosome particles per kilogram to about 10 12 virosome particles per kilogram. More preferably from about 10 8 virosome particles per kilogram to about 10 12 virosome particles per kilogram.
Alternative exemplary doses may range from about 10 4 virosome particles per gram of gland to about 10 12 virosome particles per gram of gland (e.g., about 10 4、105、106、107、108、109、1011、1012 or a range thereof).
In some aspects, the dosage is determined by the amount of fluid required to fill the gland. In order for the vector to come into contact with the cells, the glands should be filled with fluid in order to introduce the vector into the cells. In IR patients, the volume ranges from about 500 μl to about 2.5mL (e.g., ,500μL、600μL、700μL、800μL、900μL、1mL、1.1mL、1.2mL、1.3mL、1.3mL、1.4mL、1.5mL、1.6mL、1.7mL、1.8mL、1.9mL、2mL、2.1mL、2.2mL、2.3mL、2.4mL.2.5mL, or a range thereof), depending on atrophy and fibrosis.
The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
Example 1
This example shows that administration of a vector encoding AQP1 (AAV vector) before and after IR treatment promotes salivary gland function.
All murine experiments were approved by the national institute of oral and craniofacial (National Institute of DENTAL AND Craniofacial Research, NIDCR) animal care and use committee. Female C3H mice (national cancer institute, national Cancer Institute Animal Production Area) were used at 8 weeks of age. Groups of 7-8C 3H mice were treated with AAV2 vector encoding GFP, AQP1, neurturin or a combination of AAV2-AQP1+AAV2-neurturin 10 days before (IR) irradiation or 2 months after irradiation.
In particular, the treatment group included injections of AAV2-GFP (10 10 vp/g) or AAV2-hAQP1 (10 10 or 10 7 vp/g) or AAV2NRTN (10 6、108 or 10 10 vp/g) 10 days before or 60 days after IR. Baseline saliva (non-IR) was collected prior to vehicle delivery or IR. To irradiate the salivary glands, each animal was placed in a specially constructed Lucite jig. The clamp secures the animal without the use of anesthetic and allows IR only to the head and neck area.
Mice were irradiated 5 times (6 Gy/day for 5 days) using Therapax DXT X-ray radiator (Pantak). After IR, animals were removed from the jig, kept in a climate and light controlled environment (5 animals per cage) and allowed free access to food and water. For delivery of the viral vector into the submandibular glands, mice were anesthetized intramuscularly with ketamine (60 mg/kg) and xylazine (8 mg/kg), and then the vector was delivered into both submandibular glands by reverse catheter infusion. During intubation, 0.5mg/kg atropine was administered intramuscularly to inhibit salivary secretion, thereby improving transduction efficiency.
For saliva collection, mice were anesthetized as described above, and then pilocarpine at 0.25mg/kg body weight was subcutaneously injected to stimulate salivation. All saliva was collected with a 75mm hematocrit tube (Drummond) into a 1.5mL pre-weighed Eppendorf tube for 20 minutes and frozen immediately. After 10 months, mice were sacrificed in a carbon dioxide chamber and glands were removed for analysis. Saliva (baseline/non-IR) was collected from mice before the experiment started. The results are shown in fig. 1 to 3.
The results show that treatment with AAV2-AQP1 prevents loss of saliva flow before IR compared to AAV2-GFP treated mice, or that AAV2-AQP1 treatment after IR can initiate saliva flow recovery (p < 01) (see figures 1-3). In contrast, neurturin can only prevent loss of saliva flow and cannot begin substantial recovery. Furthermore, the combination of the two vectors was not synergistic and significantly increased saliva flow before or after IR beyond the levels achieved by AAV2AQP1 vector alone.
These results support that administration of AAV-AQP1 before and after IR treatment promotes salivary gland function.
Prior to the present invention, it was conventionally understood that AQP1 gene therapy required stable epithelial cells to produce an enhanced fluid movement pathway for AQP1 expression. In clinical trials of AQP1, patients were required at least 2 years after IR treatment. Due to the significant cellular renewal and remodeling following IR treatment, it is expected that AQP1AAV DNA will be lost (i.e., not sustained) from transduced cells following IR treatment and thus unable to produce an enhanced fluid movement pathway. For example, malik et al, J.Virol.,71 (3): 1776-1783 (1997)) teaches that AAV is only continuously present in non-dividing cells and lost in dividing cell populations over time. Li et al, int.j. Radiation Oncology biol.Phys.,62 (5): 1510-1516 (2005)) teaches that significant remodeling occurs in the IR posterior gland and that the alteration is followed by a loss of function. Furthermore Vitolo et al, oral Diseases,8:183-191 (2002)) teach that AQP1 gene therapy is useful for repairing glands, while other methods are useful for preventing IR damage to glands. Vitolo et al also disclose that salivary glands are a slowly dividing cell population in which AAV transduction may persist.
Thus, prior to the present invention, AQP1 gene therapy was not considered a prophylactic approach for IR-induced hypofunction, as AQP1 gene therapy was not persisted in changing and remodelling environments, such as the salivary glands after IR. However, as described herein, it is surprising and unexpected that the invention disclosed herein, administration of AAV-AQP1 prior to and after IR treatment, promotes salivary gland function.
Example 2
This example characterizes the mechanism of pre-IR and post-IR saliva flow.
Data from IR mice that were not IR, GFP-treated or single cell RNAseq before AQP1 treatment (AQP 1B) or after AQP1 treatment (AQP 1A) were used to generate UMAP and identify 16 different cell clusters. Cell distributions between different clusters under each of the 4 conditions were compared for generating the heat map of fig. 4.
AQP1B can be found in the same branches as non-IR, while GFP and AQP1A are in branches separate from each other and in AQP 1B/non-IR branches. This result suggests that the cell population is different between AQP1A and AQP 1B. Furthermore, the branched organization of AQP1B based on the distribution of cell types is most similar to non-IR, whereas AQP1A forms a different branch from this group and the IR-effector GFP group.
These results support that saliva flow is the result of gland protection if administered prior to IR; however, if given after IR, restoration of saliva flow in different cell populations and environments is possible.
Example 3
This example shows that administration of AAV-AQP1 prior to irradiation results in less pathological changes than administration of AAV-AQP1 after irradiation.
The histology of the mouse submandibular glands was assessed. Images of mice administered with AAV-GFP prior to irradiation (fig. 5A), AAV-AQP1 prior to irradiation (fig. 5B), AAV-GFP after irradiation (fig. 5C), and AAV-AQP1 after irradiation (fig. 5D) were taken from all scans of the same region of each gland typically near the door. The overall morphological changes of the glands were assessed by H & E staining.
Sections were assessed for atrophy, fibrosis and immune infiltration using a grading scale of 0 to 3. Overall, all of these samples had increased atrophy and fibrosis. They also have increased inflammation, intraglandular hair center formation, and reactive lymph nodes present within the glandular sac. The number of multinuclear acinar cells increases and the duct in partial area proliferates.
However, the average score for the post-treatment group was 2.2+/-.54 (p > 0.05) for the pre-treatment group. Thus, the post-IR treatment group tended to show more significant pathological changes than the pre-IR treatment group (fig. 5A-5D).
These results support that administration of the AQP 1-encoding vector prior to irradiation reduces the deleterious effects observed when AQP 1-encoding vectors are administered post-irradiation.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
In the context of describing the present invention (particularly in the context of the claims), the use of the terms "a" and "an" and "the" and "at least one" and similar referents are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one followed by a list of one or more items (e.g.," at least one of a and B ") should be interpreted to mean one item selected from the list of items (a or B) or any combination of two or more of the list of items (a and B), unless otherwise indicated herein or clearly contradicted by context. Unless otherwise indicated, the terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted herein. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually recited herein. 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 herein, is intended merely to better illuminate 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 any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to 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. Furthermore, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
Biological sequences
The following sequences are listed herein:
SEQ ID NO:1:
1masefkkklf wravvaefla ttlfvfisig salgfkypvg nnqtavqdnv kvslafglsi
61atlaqsvghi sgahlnpavt lglllscqis ifralmyiia qcvgaivata ilsgitsslt
121gnslgrndla dgvnsgqglg ieiigtlqlv lcvlattdrr rrdlggsapl aiglsvalgh
181llaidytgcg inparsfgsa vithnfsnhw ifwvgpfigg alavliydfi laprssdltd
241rvkvwtsgqv eeydldaddi nsrvemkpk
SEQ ID NO:2:
1mpgarplplv lvpqntlawm qldakapahp rplqllgrvg pgsrqladgv nsgqglgiei
61igtlqlvlcv lattdrrrrd lggsaplaig lsvalghlla idytgcginp arsfgsavit
121hnfsnhwifw vgpfiggala vliydfilap rssdltdrvk vwtsgqveey dldaddinsr
181vemkpk
SEQ ID NO:3:
1mfwtfgyeav spagpshlfa slllgvllti tfmpgarplp lvlvpqntla wmqldakapa
61hprplqllgr vgpgsrqlad gvnsgqglgi eiigtlqlvl cvlattdrrr rdlggsapla
121iglsvalghl laidytgcgi nparsfgsav ithnfsnhwi fwvgpfigga lavliydfil
181aprssdltdr vkvwtsgqve eydldaddin srvemkpk
SEQ ID NO:4:
1mqsgmgwnvl dfwladgvns gqglgieiig tlqlvlcvla ttdrrrrdlg gsaplaigls
61valghllaid ytgcginpar sfgsavithn fsnhwifwvg pfiggalavl iydfilaprs
121sdltdrvkvw tsgqveeydl daddinsrve mkpk
SEQ ID NO:5
1maseikkklf wravvaefla mtlfvfisig salgfnyple rnqtlvqdnv kvslafglsi
61atlaqsvghi sgahlnpavt lglllscqis ilravmyiia qcvgaivata ilsgitsslv
121dnslgrndla hgvnsgqglg ieiigtlqlv lcvlattdrr rrdlggsapl aiglsvalgh
181llaidytgcs inparsfgsa vltrnfsnhw ifwvgpfigg alavliydfi laprssdftd
241rmkvwtsgqv eeydldaddi nsrvemkpk
Claims (15)
1. A method of preventing or reducing radiation-induced saliva dysfunction in a subject, comprising:
(a) Administering to said subject a carrier encoding Aquaporin (AQP), and
(B) Administering ionizing radiation to the subject after (a),
Thereby preventing or alleviating radiation-induced saliva dysfunction in the subject.
2. Use of a carrier encoding Aquaporin (AQP) in a method of preventing or alleviating radiation-induced saliva dysfunction in a subject, wherein the method comprises:
(a) Administering to said subject a carrier encoding Aquaporin (AQP), and
(B) Administering ionizing radiation to the subject after (a),
Thereby preventing or alleviating radiation-induced saliva dysfunction in the subject.
3. The method of claim 1 or the use of claim 2, wherein the vector is administered to the salivary gland of the subject.
4. A method or use according to any one of claims 1 to 3 wherein the AQP protein comprises an AQP1 protein.
5. The method or use of claim 4, wherein said AQP1 protein comprises a human AQP1 protein.
6. The method or use of any one of claims 1-5, wherein the vector comprises a viral vector.
7. The method or use of claim 6, wherein the viral vector comprises an adenovirus vector.
8. The method or use of claim 7, wherein the adenovirus vector comprises serotype 2 or serotype 5.
9. The method or use of claim 6, wherein the viral vector comprises an adeno-associated virus (AAV) vector.
10. The method or use of claim 9, wherein the AAV vector comprises AAV2, AAV5, AAV6, AAV44.9 or BAAV.
11. The method or use of claim 9 or 10, wherein the AAV vector is administered as a virosome comprising an AAV vector.
12. The method or use of claim 11, wherein the virosomes comprise AAV virosomes.
13. The method or use according to any one of claims 1-12, wherein salivary gland function is maintained at a level equal to or at least equal to salivary gland function prior to administration of the ionizing radiation.
14. The method or use of any one of claims 1-13, wherein the subject is a human patient.
15. The method or use of claim 14, wherein the patient has head and neck cancer.
Applications Claiming Priority (4)
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US63/229,279 | 2021-08-04 | ||
US202263297342P | 2022-01-07 | 2022-01-07 | |
US63/297,342 | 2022-01-07 | ||
PCT/US2022/074559 WO2023015269A1 (en) | 2021-08-04 | 2022-08-04 | Aqp1 gene therapy to prevent radiation induced salivary hypofunction |
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CN117940147A true CN117940147A (en) | 2024-04-26 |
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CN202280058429.9A Pending CN117940147A (en) | 2021-08-04 | 2022-08-04 | AQP1 gene therapy for preventing radiation-induced hyposalivation |
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2022
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